Imidacloprid (IMI), a neonicotinoid insecticide, has potential cytotoxic and genotoxic effects on human and experimental models, respectively. While being an emerging environmental contaminant, occupational exposure and related cellular mechanisms are unknown. Herein, we were motivated by a specific patient case where occupational exposure to an IMI-containing plant protection product was associated with the diagnosis of Bell's palsy. The aim was to investigate the toxic effects and cellular mechanisms of IMI exposure on glial cells (D384 human astrocytes) and on human fibroblasts (AG01518). IMI-treated astrocytes showed a reduction in cell number and dose-dependent cytotoxicity at 24 h. Lower doses of IMI induced reactive oxygen species (ROS) and lysosomal membrane permeabilisation (LMP), causing apoptosis and autophagic dysfunction, while high doses caused significant necrotic cell death. Using normal fibroblasts, we found that IMI-induced autophagic dysfunction and lysosomal damage, activated lysophagy, and resulted in a compensatory increase in lysosomes. In conclusion, the observed IMI-induced effects on human glial cells and fibroblasts provide a possible link between IMI cytotoxicity and neurological complications observed clinically in the patient exposed to this neonicotinoid insecticide.

Hemostasis is a critical physiological process that stops bleeding at the site of an injury while ensuring normal blood flow elsewhere, thereby preventing excessive clot formation that could lead to dangerous conditions like thrombosis. This delicate balance is influenced by genetics, medical conditions such as cancer, and various medications. When a blood vessel is damaged, platelets adhere to the exposed area, become activated, and aggregate to form an initial plug. Coagulation factors, particularly thrombin, create a strong fibrin network to stabilize the clot. Disruptions in this process can result in significant bleeding or dangerous clot formation.

This thesis aims to explore and understand the factors affecting coagulation and the risks of thrombotic events in different medical contexts. This includes studying genetic variability in the protease-activated receptor 4 (PAR4) gene (specifically the Ala120Thr variant) among sub-Saharan African populations, identifying genetic and non-genetic risk factors for venous thromboembolism (VTE) in patients with the brain cancer glioblastoma multiforme (GBM), and investigating the impact of intravenous morphine on platelet activity in patients with ST-elevation myocardial infarction (STEMI) treated with ticagrelor, a P2Y12 inhibitor.  

The A allele of the rs773902 single-nucleotide polymorphism (SNP) in the PAR4 gene (F2RL3) substitutes threonine for alanine at the 120th protein position (Thr120). This allele is more prevalent in African populations compared to Caucasian populations, although previous studies did not specify the geographic ancestry of participants. Thr120 is associated with higher PAR4-induced human platelet aggregation and Ca2+ flux. Our study found that the frequency of the A allele in the Somali population is significantly lower than previously reported for African Americans. The A allele frequency in Somalis is 38%, compared to 63% for African Americans. The A allele frequency in Somalis is closer to that of the Maasai population in Kenya (41%), but vastly different from the Esan population in Nigeria (68%).  

Certain cancers, such as GBM, are associated with a higher risk of VTE, including deep vein thrombosis (DVT) and pulmonary embolism (PE). Our research identified blood group B as a significant risk factor for patients with GBM (OR=6.91; 95% CI=2.2–24.1; P =0.001). Also, GBM tumors in the frontal lobe are associated with an increased risk of VTE (OR=3.14; 95% CI=1.1–10.7; P =0.05). 

Our study on morphine, commonly used for pain management in STEMI patients, found that morphine is associated with increased platelet aggregation one hour after percutaneous coronary intervention (PCI), impacting the efficacy of ticagrelor. Morphine delays platelet inhibition by affecting the pharmacodynamics of antiplatelet therapy, likely by delaying gastric emptying. However, this effect is short-lived, as platelet reactivity returns to similar levels in both groups 12 hours post-PCI. Despite this immediate impact on platelet function, our research found no significant differences in biomarkers of platelet activity, coagulation, or inflammation between the morphine and non-morphine groups. Additionally, all patients in our study were administered unfractionated heparin injections or bivalirudin infusion during primary PCI, which may help control the risk of blood clot formation.  

These studies collectively emphasize the need for individualized strategies to manage thrombotic risks and coagulation. The significant genetic variability among sub-Saharan African populations highlights the need for precise genetic research to understand how genetics influence coagulation and develop personalized medical strategies. The increased risk of cancer-associated thrombosis, particularly in patients with GBM, calls for individualized anticoagulant therapies based on unique risk profiles, such as blood group typing and tumor location. Incorporating these insights into clinical practice can help healthcare providers better identify high-risk patients and tailor thromboprophylaxis strategies accordingly. Similarly, the impact of morphine on patients with STEMI treated with ticagrelor requires careful consideration.  

In conclusion, these findings underscore the importance of a personalized approach in managing coagulation and thrombotic risks. The studies show that genetic variability, specific medical conditions, and medication effects are crucial in thrombotic risk. Therefore, customized strategies based on individual patient profiles and contexts are essential for effectively managing and preventing thrombotic events. 

ST-segment elevation myocardial infarction (STEMI) is usually caused by a ruptured atherosclerotic plaque, with subsequent thrombus formation. Platelet inhibition and primary percutaneous coronary intervention (PCI) are essential treatments. Morphine, used to relieve pain and anxiety in STEMI patients, delays the onset of P2Y12 inhibitors. This study aimed to further explore the association between platelet activity and morphine treatment in patients with STEMI. In this sub-study of the VALIDATE-SWEDHEART trial, 89 STEMI patients treated with ticagrelor, and primary PCI were included. Platelet aggregation and biomarkers of platelet activity, coagulation, and inflammation (sP-selectin, thrombin-antithrombin complexes, prothrombin fragments 1 + 2, CD40L, CRP, beta-thromboglobulin, and pentraxin3) were assessed at three time points: before, one, and twelve hours after PCI. Of the 89 patients, 40 received morphine before hospital arrival. There were no significant differences in age, sex, medical history, or coronary disease extent. One hour after PCI, ADP-induced (36 vs 61, p < .001), arachidonic acid-induced (20 vs 36, p = .003), collagen-induced (48 vs 60, p = .03) aggregation, and the proportion of high on-treatment ADP-induced platelet reactivity (27% vs 60%, p = .001) were significantly higher in morphine-treated patients. No significant differences were found before or 12 hours after PCI. No significant differences in platelet activity biomarkers were observed. Morphine increased platelet aggregation in STEMI patients but did not affect biomarkers.

The aim of this study was to investigate whether CD74 levels in atherosclerotic lesions are associated with inflammation, apoptosis, plaque severity, and clinical symptoms among patients with carotid atherosclerosis. We further studied whether CD74 expression is associated with apoptosis in macrophages induced by 7ketocholesterol (7keto). Sixty-one carotid samples (39 males and 22 females) were immunostained with macrophages, smooth muscle cells, CD74, ferritin, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling), and thrombin receptors. Double immunocytochemistry of CD74 and caspase 3 or CD74 and Annexin V was performed on THP-1 macrophages exposed to 7keto. In human carotid plaques, CD74 expression is lesion-dependently increased and is associated with necrotic core formation and plaque rupture, clinical symptoms, macrophage apoptosis, ferritin, and thrombin receptors. CD74 levels were inversely correlated to high-density lipoproteins and statin treatment, and positively correlated to triglycerides. In THP-1 macrophages, 7keto induced a significant increase in levels of CD74, ferritin, and apoptotic cell death. This study suggests that CD74 in apoptotic macrophages is linked to inflammation and thrombosis in progression of human atherosclerotic plaques, lipid metabolism, and clinical manifestation in atherosclerosis. Surface CD74 in apoptotic macrophages and ferritin production induced by oxidized lipids may contribute to inflammation and plaque vulnerability in atherosclerosis.

Hepcidin is upregulated by increased body iron stores and inflammatory cytokines. It is associated with cardiovascular events, arterial stiffness, and increased iron accumulation in human atheroma with hemorrhage. However, it is unknown whether the expression of hepcidin in human carotid plaques is related to plaque severity and whether hepcidin expression differs between men and women. Carotid samples from 58 patients (38 males and 20 females) were immunostained with hepcidin, macrophages, ferritin, and transferrin receptor. Immunocytochemistry of hepcidin was performed on THP-1 macrophages exposed to iron or 7betahydroxycholesterol. Hepcidin expression significantly increases with the progression of human atherosclerotic plaques. Plaques of male patients have significantly higher levels of hepcidin. Expressions of hepcidin are significantly correlated with the accumulation of CD68-positive macrophages and transferrin receptor 1 (TfR1) and apoptosis. In vitro, hepcidin is significantly increased in macrophages exposed to iron and moderately increased following 7-oxysterol treatment. In the cultured cells, suppression of hepcidin protected against macrophage cell death, lysosomal membrane permeabilization, and oxidative stress. Hepcidin may play a crucial role in the development and progression of atherosclerosis. The differential expression of hepcidin in male and female patients and its significant correlations with plaque severity, highlight the potential of hepcidin as a biomarker for risk stratification and therapeutic targeting in atherosclerosis.