Nile Red Quantifier:A novel and quantitative tool to study lipid accumulation in patient-derived circulating monocytes using confocal microscopy

The inflammatory profile of circulating monocytes is an important biomarker for atherosclerotic plaque vulnerability. Recent research revealed that peripheral lipid uptake by monocytes alters their phenotype toward an inflammatory state and this coincides with an increased lipid droplet (LD) content. Determination of lipid content of circulating monocytes is, however, not very well established. Based on Nile Red (NR) neutral LD imaging, using confocal microscopy and computational analysis, we developed NR Quantifier (NRQ), a novel quantification method to assess LD content in monocytes. Circulating monocytes were isolated from blood and used for the NR staining procedure. In monocytes stained with NR, we clearly distinguished, based on 3D imaging, phospholipids and exclusively intracellular neutral lipids. Next, we developed and validated NRQ, a semi-automated quantification program that detects alterations in lipid accumulation. NRQ was able to detect LD alterations after ex vivo exposure of isolated monocytes to freshly isolated LDL in a time-and dose-dependent fashion. Finally, we validated NRQ in patients with familial hypercholesterolemia and obese subjects in pre- and postprandial state. In conclusion, NRQ is a suitable tool to detect even small differences in neutral LD content in circulating monocytes using NR staining

Severe autoimmune hemolytic anemia; epidemiology, clinical management, outcomes and knowledge gaps

Autoimmune hemolytic anemia (AIHA) is an acquired hemolytic disorder, mediated by auto-antibodies, and has a variable clinical course ranging from fully compensated low grade hemolysis to severe life-threatening cases. The rarity, heterogeneity and incomplete understanding of severe AIHA complicate the recognition and management of severe cases. In this review, we describe how severe AIHA can be defined and what is currently known of the severity and outcome of AIHA. There are no validated predictors for severe clinical course, but certain risk factors for poor outcomes (hospitalisation, transfusion need and mortality) can aid in recognizing severe cases. Some serological subtypes of AIHA (warm AIHA with complement positive DAT, mixed, atypical) are associated with lower hemoglobin levels, higher transfusion need and mortality. Currently, there is no evidence-based therapeutic approach for severe AIHA. We provide a general approach for the management of severe AIHA patients, incorporating monitoring, supportive measures and therapeutic options based on expert opinion. In cases where steroids fail, there is a lack of rapidly effective therapeutic options. In this era, numerous novel therapies are emerging for AIHA, including novel complement inhibitors, such as sutimlimab. Their potential in severe AIHA is discussed. Future research efforts are needed to gain a clearer picture of severe AIHA and develop prediction models for severe disease course. It is crucial to incorporate not only clinical characteristics but also biomarkers that are associated with pathophysiological differences and severity, to enhance the accuracy of prediction models and facilitate the selection of the optimal therapeutic approach. Future clinical trials should prioritize the inclusion of severe AIHA patients, particularly in the quest for rapidly acting novel agents

Carotid arterial wall inflammation in peripheral artery disease is augmented by type 2 diabetes: a cross-sectional study

Patients with peripheral artery disease (PAD) are at increased risk of secondary events, which is exaggerated in the presence of type 2 diabetes mellitus. Diabetes is associated with a systemic pro-inflammatory state. We therefore investigated the cumulative impact of PAD and type 2 diabetes on carotid arterial wall inflammation. As recent data suggest a detrimental role of exogenous insulin on cardiovascular disease, we also included a group of insulin users. (18)F-fluorodeoxyglucose positron emission tomography with computed tomography ((18)F-FDG PET/CT) imaging showed increased carotid arterial wall inflammation, assessed as target-to-background ratio (TBR), in PAD patients without diabetes (PAD-only: n = 11, 1.97 ± 0.57) compared with matched controls (n = 12, 1.49 ± 0.57; p = 0.009), with a significant further TBR increase in PAD patients with type 2 diabetes (PAD-DM, n = 23, 2.90 ± 1, p = 0.033 vs PAD-only). TBR of insulin users (n = 12, 3.31 ± 1.14) was higher compared with patients on oral medication only (n = 11, 2.44 ± 0.76, p = 0.035), despite comparable PAD severity (Fontaine stages), BMI and CRP. Multivariate regression analysis showed that Hba1c and plasma insulin levels, but not dose of exogenous insulin, correlated with TBR. Concurrent diabetes significantly augments carotid arterial wall inflammation in PAD patients. A further increase in those requiring insulin was observed, which was associated with diabetes severity, rather than with the use of exogenous insulin itsel

Increased arterial wall inflammation in patients with ankylosing spondylitis is reduced by statin therapy

Ankylosing spondylitis (AS) is a chronic inflammatory disease with involvement of axial and sacroiliac joints. In addition, patients with AS have increased risk of cardiovascular disease (CVD), which might be attributed to enhanced inflammatory activity of the arterial wall. In the present study, we compared the level of carotid arterial wall inflammation in patients with AS with healthy controls using (18)F-fluorodeoxyglucose ((18)F-FDG) positron emission tomography with CT. As arterial wall inflammation is reduced by statin therapy, we subsequently assessed the effect of 3-month statin therapy on arterial wall inflammation in AS. We included 24 patients with AS (age 44±10, 72% males) without a history of CVD and 20 controls matched for age and gender. Patients with AS had lower high-density lipoprotein cholesterol and increased C reactive protein (CRP) compared with controls. The 10-year CVD risk was 2% in both groups. Notwithstanding, patients with AS had a 20% increase in arterial wall (18)F-FDG uptake compared with controls. Three-month atorvastatin 40 mg daily significantly lowered low-density lipoprotein cholesterol (baseline 3.55±1.15 mmol/L, -53%) and CRP (baseline 5.0 (1.5-9.3) mg/L, -58%) with a concomitant decrease of carotid arterial wall inflammation (maximum target-to-background ratio from 1.90±0.30 to 1.67±0.27; p=0.009). Patients with AS and without other CVD risk factors have increased arterial wall inflammation, which decreases upon statin therapy. These subjects are not identified as being at risk in current cardiovascular prevention guidelines. Our data support the need to revise CV disease management in AS, with perhaps a role for early statin therap