Blockade of the CD47/SIRPα checkpoint axis potentiates the macrophage-mediated anti-tumor efficacy of tafasitamab.

Macrophages are one of the key mediators of the therapeutic effects exerted by monoclonal antibodies, such as the anti-CD19 antibody tafasitamab, approved in combination with lenalidomide for the treatment of relapsed or refractory (r/r) diffuse large B cell lymphoma (DLBCL). However, antibody-dependent cellular phagocytosis (ADCP) in the tumor microenvironment can be counteracted by increased expression of the inhibitory receptor SIRPα on macrophages and its ligand, the immune checkpoint molecule CD47 on tumor cells. The aim of this study was to investigate the impact of the CD47-SIRPα axis on tafasitamabmediated phagocytosis and explore the potential of anti-CD47 blockade to enhance its antitumor activity. Elevated expression of both SIRPα and CD47 was observed in DLBCL patient-derived lymph node biopsies compared to healthy controls. CRISPR-mediated CD47 overexpression impacted tafasitamab-mediated ADCP in vitro and increased expression of SIRPα on macrophages correlated with decreased ADCP activity of tafasitamab against DLBCL cell lines. Combination of tafasitamab and an anti-CD47 blocking antibody enhanced ADCP activity of in vitro generated macrophages. Importantly, tafasitamab-mediated phagocytosis was elevated in combination with CD47 blockade using primary DLBCL cells and patient-derived lymphoma-associated macrophages (LAMs) in an autologous setting. Furthermore, lymphoma cells with low CD19 expression were efficiently eliminated by the combination treatment. Finally, combined treatment of tafasitamab and an anti-CD47 antibody resulted in enhanced tumor volume reduction and survival benefit in lymphoma xenograft mouse models. These findings provide evidence that CD47 blockade can enhance the phagocytic potential of tumor targeting immunotherapies such as tafasitamab and suggest there is value in exploring the combination in the clinic.

Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding.

Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

SAR296968, a Novel Selective Na+/Ca2+ Exchanger Inhibitor, Improves Ca2+ Handling and Contractile Function in Human Atrial Cardiomyocytes.

BACKGROUND: In reverse-mode, cardiac sodium-calcium exchanger (NCX) can increase the cytoplasmic Ca2+ concentration in response to high intracellular Na+ levels, which may contribute to diastolic contractile dysfunction. Furthermore, increased spontaneous Ca2+ release from intracellular stores can activate forward mode NCX. The resulting transient inward current causes delayed afterdepolarization (DAD)-dependent arrhythmias. Moreover, recently, NCX has been associated with impaired relaxation and reduced cardiac function in heart failure with preserved ejection fraction (HFpEF). Since NCX is upregulated in human chronic atrial fibrillation (AF) as well as heart failure (HF), specific inhibition may have therapeutic potential. OBJECTIVE: We tested the antiarrhythmic, lusitropic and inotropic effects of a novel selective NCX-inhibitor (SAR296968) in human atrial myocardium. METHODS AND RESULTS: Right atrial appendage biopsies of 46 patients undergoing elective cardiac surgery in a predominant HFpEF cohort (n = 24/46) were investigated. In isolated human atrial cardiomyocytes, SAR296968 reduced the frequency of spontaneous SR Ca2+ release events and increased caffeine transient amplitude. In accordance, in isolated atrial trabeculae, SAR296968 enhanced the developed tension after a 30 s pause of electrical stimulation consistent with reduced diastolic sarcoplasmic reticulum (SR) Ca2+ leak. Moreover, compared to vehicle, SAR296968 decreased steady-state diastolic tension (at 1 Hz) without impairing developed systolic tension. Importantly, SAR296968 did not affect the safety parameters, such as resting membrane potential or action potential duration as measured by patch clamp. CONCLUSION: The novel selective NCX-inhibitor SAR296968 inhibits atrial pro-arrhythmic activity and improves diastolic and contractile function in human atrial myocardium, which may have therapeutic implications, especially for treatment of HFpEF.

Enhanced Cardiac CaMKII Oxidation and CaMKII-Dependent SR Ca Leak in Patients with Sleep-Disordered Breathing.

BACKGROUND: Sleep-disordered breathing (SDB) is associated with increased oxidant generation. Oxidized Ca/calmodulin kinase II (CaMKII) can contribute to atrial arrhythmias by the stimulation of sarcoplasmic reticulum Ca release events, i.e., Ca sparks. METHODS: We prospectively enrolled 39 patients undergoing cardiac surgery to screen for SDB and collected right atrial appendage biopsies. RESULTS: SDB was diagnosed in 14 patients (36%). SDB patients had significantly increased levels of oxidized and activated CaMKII (assessed by Western blotting/specific pulldown). Moreover, SDB patients showed a significant increase in Ca spark frequency (CaSpF measured by confocal microscopy) compared with control subjects. CaSpF was 3.58 ± 0.75 (SDB) vs. 2.49 ± 0.84 (no SDB) 1/100 µm-1s-1 (p < 0.05). In linear multivariable regression models, SDB severity was independently associated with increased CaSpF (B [95%CI]: 0.05 [0.03; 0.07], p < 0.001) after adjusting for important comorbidities. Interestingly, 30 min exposure to the CaMKII inhibitor autocamtide-2 related autoinhibitory peptide normalized the increased CaSpF and eliminated the association between SDB and CaSpF (B [95%CI]: 0.01 [-0.1; 0.03], p = 0.387). CONCLUSIONS: Patients with SDB have increased CaMKII oxidation/activation and increased CaMKII-dependent CaSpF in the atrial myocardium, independent of major clinical confounders, which may be a novel target for treatment of atrial arrhythmias in SDB.

AA amyloid fibrils from diseased tissue are structurally different from in vitro formed SAA fibrils.

Systemic AA amyloidosis is a world-wide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from serum amyloid A (SAA) protein. Using cryo electron microscopy we here show that amyloid fibrils which were purified from AA amyloidotic mice are structurally different from fibrils formed from recombinant SAA protein in vitro. Ex vivo amyloid fibrils consist of fibril proteins that contain more residues within their ordered parts and possess a higher ß-sheet content than in vitro fibril proteins. They are also more resistant to proteolysis than their in vitro formed counterparts. These data suggest that pathogenic amyloid fibrils may originate from proteolytic selection, allowing specific fibril morphologies to proliferate and to cause damage to the surrounding tissue.

QCM-arrays for sensing terpenes in fresh and dried herbs via bio-mimetic MIP layers.

A piezoelectric 10 MHz multichannel quartz crystal microbalance (MQCM), coated with six molecularly imprinted polystyrene artificial recognition membranes have been developed for selective quantification of terpenes emanated from fresh and dried Lamiaceae family species, i.e., rosemary (Rosmarinus Officinalis L.), basil (Ocimum Basilicum) and sage (Salvia Officinalis). Optimal e-nose parameters, such as layer heights (1-6 KHz), sensitivity <20 ppm of analytes, selectivity at 50 ppm of terpenes, repeatability and reproducibility were thoroughly adjusted prior to online monitoring. Linearity in reversible responses over a wide concentration range <20-250 ppm has been achieved. Discrimination between molecules of similar molar masses, even for isomers, e.g. α-pinene and ß-pinene is possible. The array has proven its sensitive and selective properties of sensor responses (20-1,200 Hz) for the difference of fresh and dried herbs. The sensor data attained was validated by GC-MS, to analyze the profiles of sensor emanation patterns. The shelf-life of herbs was monitored via emanation of organic volatiles during a few days. Such an array in association with data analysis tools can be utilized for characterizing complex mixtures.

The oral Ca/calmodulin-dependent kinase II inhibitor RA608 improves contractile function and prevents arrhythmias in heart failure.

AIMS: Excessive activation of Ca/calmodulin-dependent kinase II (CaMKII) is of critical importance in heart failure (HF) and atrial fibrillation. Unfortunately, lack of selectivity, specificity, and bioavailability have slowed down development of inhibitors for clinical use. We investigated a novel CaMKIIδ/CaMKIIÉ£-selective, ATP-competitive, orally available CaMKII inhibitor (RA608) on right atrial biopsies of 119 patients undergoing heart surgery. Furthermore, we evaluated its oral efficacy to prevent deterioration of HF in mice after transverse aortic constriction (TAC). METHODS AND RESULTS: In human atrial cardiomyocytes and trabeculae, respectively, RA608 significantly reduced sarcoplasmic reticulum Ca leak, reduced diastolic tension, and increased sarcoplasmic reticulum Ca content. Patch-clamp recordings confirmed the safety of RA608 in human cardiomyocytes. C57BL6/J mice were subjected to TAC, and left ventricular function was monitored by echocardiography. Two weeks after TAC, RA608 was administered by oral gavage for 7 days. Oral RA608 treatment prevented deterioration of ejection fraction. At 3 weeks after TAC, ejection fraction was 46.1 ± 3.7% (RA608) vs. 34.9 ± 2.6% (vehicle), n = 9 vs. n = 12, P < 0.05, ANOVA, which correlated with significantly less CaMKII autophosphorylation at threonine 287. Moreover, a single oral dose significantly reduced inducibility of atrial and ventricular arrhythmias in CaMKIIδ transgenic mice 4 h after administration. Atrial fibrillation was induced in 6/6 mice for vehicle vs. 1/7 for RA608, P < 0.05, 'n - 1' χ2 test. Ventricular tachycardia was induced in 6/7 for vehicle vs. 2/7 for RA608, P < 0.05, 'n - 1' χ2 test. CONCLUSIONS: RA608 is the first orally administrable CaMKII inhibitor with potent efficacy in human myocytes. Moreover, oral administration potently inhibits arrhythmogenesis and attenuates HF development in mice in vivo.

MDCT assessment of airway wall thickness in COPD patients using a new method: correlations with pulmonary function tests.

Quantitative assessment of airway-wall dimensions by computed tomography (CT) has proven to be a marker of airway-wall remodelling in chronic obstructive pulmonary disease (COPD) patients. The objective was to correlate the wall thickness of large and small airways with functional parameters of airflow obstruction in COPD patients on multi-detector (MD) CT images using a new quantification procedure from a three-dimensional (3D) approach of the bronchial tree. In 31 patients (smokers/COPD, non-smokers/controls), we quantitatively assessed contiguous MDCT cross-sections reconstructed orthogonally along the airway axis, taking the point-spread function into account to circumvent over-estimation. Wall thickness and wall percentage were measured and the per-patient mean/median correlated with FEV1 and FEV1%. A median of 619 orthogonal airway locations was assessed per patient. Mean wall percentage/mean wall thickness/median wall thickness in non-smokers (29.6%/0.69 mm/0.37 mm) was significantly different from the COPD group (38.9%/0.83 mm/0.54 mm). Correlation coefficients (r) between FEV1 or FEV1% predicted and intra-individual means of the wall percentage were -0.569 and -0.560, respectively, with p < 0.001. Depending on the parameter, they were increased for airways of 4 mm and smaller in total diameter, being -0.621 (FEV1) and -0.537 (FEV1%) with p < 0.002. The wall thickness was significantly higher in smokers than in non-smokers. In COPD patients, the wall thickness measured as a mean for a given patient correlated with the values of FEV1 and FEV1% predicted. Correlation with FEV1 was higher when only small airways were considered.

Functional analysis in single-lung transplant recipients: a comparative study of high-resolution CT, 3He-MRI, and pulmonary function tests.

OBJECTIVE: To develop and evaluate a postprocessing tool to quantify ventilated split-lung volumes on the basis of (3)He-MRI and to apply it in patients after single-lung transplantation (SLTX). High-resolution CT (HRCT) was employed as a reference modality providing split air-filled lung volumes. Lung volumes derived from pulmonary function test results served as clinical parameters and were used as the "gold standard." MATERIAL AND METHODS: Eight patients (mean age, 54 years) with emphysema and six patients (mean age, 58 years) with idiopathic pulmonary fibrosis. All patients were evaluated following SLTX. HRCT was performed during inspiration (slice thickness, 1 mm; increment, 10 mm). For correlation with (3)He-MRI, HRCT images were reconstructed in coronal orientation to match the same anatomic levels. Aerated lung was determined by threshold-based segmentation of CT. (3)He-MRI was performed on a 1.5-T scanner using a two-dimensional, fast low-angle shot sequence in coronal orientation covering the whole lung after inhalation of a 300-mL bolus of hyperpolarized (3)He gas followed by normal room air for the rest of the tidal volume. Lung segmentation on (3)He-MRI was done using different thresholds. RESULTS: In emphysematous patients, (3)He-MRI showed excellent correlation (r = 0.9) with vital capacity, while CT correlated (r = 0.8) with total lung capacity. (3)He-MRI correlated well with CT (r > 0.8) for grafts and native fibrotic lungs. In emphysematous lungs, MRI showed a good correlation (r = 0.7) with the nonemphysematous lung volume from CT. Increasing thresholds in (3)He-MRI reveal differences between aerated and ventilated lung areas with a different distribution in emphysema and fibrosis. CONCLUSIONS: (3)He-MRI is superior to CT in emphysema to demonstrate ventilated lung areas that participate in gas exchange. In fibrosis, (3)He-MRI and CT have a similar impact. The decrease pattern and the intraindividual ratio between ventilation of native and transplanted lungs will have to be investigated as a new surrogate for the ventilatory follow-up in patients undergoing SLTX.

Functional evaluation of emphysema using diffusion-weighted 3Helium-magnetic resonance imaging, high-resolution computed tomography, and lung function tests.

PURPOSE: To assess the emphysematous enlargement of distal airspaces and concomitant large and small airway disease using diffusion-weighted Helium-magnetic resonance imaging (MRI), high-resolution computed tomography (HRCT), and lung function tests (LFT). METHODS: Seven patients were examined after single lung transplantation (LTx) and 1 before double LTx for various forms of emphysema. Five patients after double LTx served as controls. Patients were assessed by Helium-MRI (apparent diffusion coefficient [ADC]), HRCT (mean lung density [MLD], emphysema index [EI]), and LFT. RESULTS: Transplanted lungs: mean ADC = 0.17 cm/s, MLD = -848 H, EI = 22%. Emphysematous lungs: mean ADC = 0.33 cm/s, MLD = -922 H; EI = 54%. Good correlations were found between ADC and MLD (r = 0.6), EI (r = 0.8), intrathoracic gas volume (r = 0.7), forced expiratory volume in 1 second (r = 0.7), and forced expiratory flows (r = 0.7). In contrast, HRCT only provided moderate correlations with LFT (EI: r = 0.5; MLD: r [le] 0.4). CONCLUSION: In this initial study, He-MRI yield good correlations with HRCT and agrees better than HRCT with the functional characterization of emphysema regarding hyperinflation, large and small airway disease as provided by LFT.

Oxygen-sensitive 3He-MRI in bronchiolitis obliterans after lung transplantation.

Oxygen-sensitive 3He-MRI was studied for the detection of differences in intrapulmonary oxygen partial pressure (pO2) between patients with normal lung transplants and those with bronchiolitis obliterans syndrome (BOS). Using software developed in-house, oxygen-sensitive 3He-MRI datasets from patients with normal lung grafts (n = 8) and with BOS (n = 6) were evaluated quantitatively. Datasets were acqiured on a 1.5-T system using a spoiled gradient echo pulse sequence. Underlying diseases were pulmonary emphysema (n = 10 datasets) and fibrosis (n = 4). BOS status was verified by pulmonary function tests. Additionally, 3He-MRI was assessed blindedly for ventilation defects. Median intrapulmonary pO2 in patients with normal lung grafts was 146 mbar compared with 108 mbar in patients with BOS. Homogeneity of pO2 distribution was greater in normal grafts (standard deviation pO2 34 versus 43 mbar). Median oxygen decrease rate during breath hold was higher in unaffected patients (-1.75 mbar/s versus -0.38 mbar/s). Normal grafts showed fewer ventilation defects (5% versus 28%, medians). Oxygen-sensitive 3He-MRI appears capable of demonstrating differences of intrapulmonary pO2 between normal lung grafts and grafts affected by BOS. Oxygen-sensitive 3He-MRI may add helpful regional information to other diagnostic techniques for the assessment and follow-up of lung transplant recipients.

Respiratory lumenal change of the pharynx and trachea in normal subjects and COPD patients: assessment by cine-MRI.

The purpose of this study was to use cine-MRI during continuous respiration to measure the respiratory lumenal diameter change in the pharynx and at an upper tracheal level. Fifteen non-smokers and 23 chronic obstructive pulmonary disease (COPD) patients with smoking history (median 50 pack-years) were included. Cine-MRI with seven frames/s was performed during continuous respiration. Minimal and maximal cross-sectional lumenal diameters within the pharynx and the upper tracheal lumen area were measured. The median diameter change in the pharynx (tracheal area) was 70% (1.4 cm(2)) in volunteers and 76% (1.7 cm(2)) in smokers (P=0.98, P=0.04). Tracheal lumenal collapse was a median of 43% in volunteers and 64% in smokers (P=0.011). No clear disease-related difference of the pharynx-lumen was found. The maximal cross-sectional area of the upper trachea lumen as well as the respiratory collapse was larger in COPD patients than in normal subjects. This information is important for the modelling of ventilation and prediction of drug deposition, which are influenced by the airway diameter.

(3)He-MRI in follow-up of lung transplant recipients.

The aim of this study was to evaluate the possible contribution of (3)He-MRI to detect obliterative bronchiolitis (OB) in the follow-up of lung transplant recipients. Nine single- and double-lung transplanted patients were studied by an initial and a follow-up (3)He-MRI study. Images were evaluated subjectively by estimation of ventilation defect area and quantitatively by individually adapted threshold segmentation and subsequent calculation of ventilated lung volume. Bronchiolitis obliterans syndrome (BOS) was diagnosed using pulmonary function tests. At (3)He-MRI, OB was suspected if ventilated lung volume had decreased by 10% or more at the follow-up MRI study compared with the initial study. General accordance between pulmonary function testing and (3)He-MRI was good, although subjective evaluation of (3)He-MRI underestimated improvement in ventilation as obtained by pulmonary function tests. The (3)He-MRI indicated OB in 6 cases. According to pulmonary function tests, BOS was diagnosed in 5 cases. All diagnoses of BOS were also detected by (3)He-MRI. In 2 of these 5 cases, (3)He-MRI indicated OB earlier than pulmonary function tests. The results support the hypothesis that (3)He-MRI may be sensitive for early detection of OB and emphasize the need for larger prospective follow-up studies.