Cross-immunity against SARS-COV-2 variants of concern in naturally infected critically ill COVID-19 patients.

Critically ill patients infected with SARS-CoV-2 display adaptive immunity, but it is unknown if they develop cross-reactivity to variants of concern (VOCs). We profiled cross-immunity against SARS-CoV-2 VOCs in naturally infected, non-vaccinated, critically ill COVID-19 patients. Wave-1 patients (wild-type infection) were similar in demographics to Wave-3 patients (wild-type/alpha infection), but Wave-3 patients had higher illness severity. Wave-1 patients developed increasing neutralizing antibodies to all variants, as did patients during Wave-3. Wave-3 patients, when compared to Wave-1, developed more robust antibody responses, particularly for wild-type, alpha, beta and delta variants. Within Wave-3, neutralizing antibodies were significantly less to beta and gamma VOCs, as compared to wild-type, alpha and delta. Patients previously diagnosed with cancer or chronic obstructive pulmonary disease had significantly fewer neutralizing antibodies. Naturally infected ICU patients developed adaptive responses to all VOCs, with greater responses in those patients more likely to be infected with the alpha variant, versus wild-type.

The profile of platelet α-granule released molecules affects postoperative liver regeneration.

UNLABELLED: Platelets promote liver regeneration through site-specific serotonin release from dense granules, triggering proliferative signaling in hepatocytes. However, the effects of factors derived from platelet α-granules on liver regeneration are unclear, because α-granules contain bioactive molecules with opposing functions. Because α-granule molecules are stored in separate compartments, it has been suggested that platelets selectively release their α-granule content dependent on the environmental stimulus. Therefore, we investigated the pattern of circulating α-granule molecules during liver regeneration in 157 patients undergoing partial hepatectomy. We measured plasma levels of α-granule-derived factors in the liver vein at the end of liver resection, as well as on the first postoperative day. We observed a rapid accumulation of platelets within the liver after induction of liver regeneration. Platelet count and P-selectin (a ubiquitous cargo of α-granules) were not associated with postoperative liver dysfunction. However, low plasma levels of vascular endothelial growth factor (VEGF), but high levels of thrombospondin 1 (TSP-1), predicted liver dysfunction after resection. Patients with an unfavorable postoperative α-granule release profile (high TSP-1/low VEGF) showed substantially worse postoperative clinical outcomes. The unfavorable postoperative α-granule release profile was associated with increased postoperative portal venous pressure and von Willebrand factor antigen levels as a marker for intrahepatic endothelial dysfunction. CONCLUSION: The postoperative profile of circulating platelet-derived factors correlates with the ability of the remnant liver to regenerate. Portal venous pressure and intrahepatic endothelial dysfunction might account for the selective granule release profile. Selective modulation of platelet α-granule release in patients may represent an attractive target for therapeutic interventions to improve liver regeneration and clinical outcomes after partial hepatectomy.

Aspirin and P2Y12 Inhibitors in platelet-mediated activation of neutrophils and monocytes.

Platelets are key players in haemostasis and represent a pivotal link between inflammation, immunity and atherogenesis. Depending on the (patho)physiological environment platelets modulate various leukocyte functions via release of inflammatory mediators and direct cell-cell interactions. Elevated levels of circulating platelet-leukocyte aggregates are found in patients suffering from several thrombotic or inflammatory conditions. Platelet-monocyte and platelet-neutrophil interaction can trigger pro- and anti-inflammatory responses and modulate effector functions of all leukocyte subpopulations. These platelet-mediated immune responses have implications for the progression of cardiovascular diseases and also play a crucial role during infections, cancer, transplantations and other inflammatory diseases of several organs. Antiplatelet therapy including the COX inhibitor aspirin and/or ADP receptor P2Y12 inhibitors such as clopidogrel, prasugrel and ticagrelor are the therapy of choice for various cardiovascular complications. Both aspirin and P2Y12 inhibitors attenuate platelet-leukocyte interactions, thereby also modulating immune responses. This may have beneficial effects in some pathological conditions, while it might be detrimental in others. This review aims to summarise the current knowledge on platelet-leukocyte interactions and the impact of aspirin and P2Y12 inhibition on platelet-mediated immune responses and to give an overview on the effects of antiplatelet therapy on platelet-leukocyte interplay in various diseases.

Human Cytomegalovirus Up-Regulates Endothelin Receptor Type B: Implication for Vasculopathies?

Background. Both endothelin receptor type B ([ETBR], a G protein-coupled receptor that mediates the vascular effects of the potent vasoconstrictor endothelin-1) and human cytomegalovirus ([HCMV], a ubiquitous herpesvirus) have been implicated in the pathogenesis of cardiovascular disease (CVD). The effects of HCMV infection on ETBR expression are unknown. We hypothesized that HCMV may contribute to the pathogenesis of CVD via ETBR modulation. Methods. Human CMV effects on ETBR were studied in vitro in endothelial cells (ECs) and smooth muscle cells (SMCs) and ex vivo in human carotid plaque tissue specimens. Expression of ETBR and viral immediate-early were quantified using quantitative polymerase chain reaction. Functional consequences after ETBR blockade in ECs were examined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide proliferation, wound healing, tube formation, and flow adhesion assays. Results. Human CMV is capable of upregulating both ETBR mRNA and protein expression in ECs and SMCs. The ETBR was also abundantly expressed in ECs, foam cells, and SMCs, and, more importantly, in HCMV-positive cells in human carotid plaques. Endothelin receptor type B blockade led to decreased proliferation and reduced tumor necrosis factor α-mediated leukocyte recruitment in both uninfected and HCMV-infected ECs. Direct HCMV infection was antimigratory and antiangiogenic in ECs. Conclusions. Human CMV may contribute to CVD via ETBR induction.

Platelets mediate oxidized low-density lipoprotein-induced monocyte extravasation and foam cell formation.

OBJECTIVE: A growing body of evidence indicates that platelets contribute to the onset and progression of atherosclerosis by modulating immune responses. We aimed to elucidate the effects of oxidized low-density lipoprotein (OxLDL) on platelet-monocyte interactions and the consequences of these interactions on platelet phagocytosis, chemokine release, monocyte extravasation, and foam cell formation. APPROACH AND RESULTS: Confocal microscopy and flow cytometric analysis revealed that in vitro and in vivo stimulation with OxLDL resulted in rapid formation of platelet-monocyte aggregates, with a preference for CD16+ monocyte subsets. This platelet-monocyte interaction facilitated OxLDL uptake by monocytes, in a process that involved platelet CD36-OxLDL interaction, release of chemokines, such as CXC motif ligand 4, direct platelet-monocyte interaction, and phagocytosis of platelets. Inhibition of cyclooxygenase with acetylsalicylic acid and antagonists of ADP receptors, P2Y1 and P2Y12, partly abrogated OxLDL-induced platelet-monocyte aggregates and platelet-mediated lipid uptake in monocytes. Platelets also enhanced OxLDL-induced monocyte transmigration across an endothelial monolayer via direct interaction with monocytes in a transwell assay. Importantly, in LDLR(-/-) mice, platelet depletion resulted in a significant decrease of peritoneal macrophage recruitment and foam cell formation in a thioglycollate-elicited peritonitis model. In platelet-depleted wild-type mice, transfusion of ex vivo OxLDL-stimulated platelets induced monocyte extravasation to a higher extent when compared with resting platelets. CONCLUSIONS: Our results on OxLDL-mediated platelet-monocyte aggregate formation, which promoted phenotypic changes in monocytes, monocyte extravasation and enhanced foam cell formation in vitro and in vivo, provide a novel mechanism for how platelets potentiate key steps of atherosclerotic plaque development and plaque destabilization.

Native high density lipoproteins (HDL) interfere with platelet activation induced by oxidized low density lipoproteins (OxLDL).

Platelets and lipoproteins play a crucial role in atherogenesis, in part by their ability to modulate inflammation and oxidative stress. While oxidized low density lipoproteins (OxLDL) play a central role in the development of this disease, high density lipoproteins (HDL) represent an atheroprotective factor of utmost importance. As platelet function is remarkably sensitive to the influence of plasma lipoproteins, it was the aim of this study to clarify if HDL are able to counteract the stimulating effects of OxLDL with special emphasis on aspects of platelet function that are relevant to inflammation. Therefore, HDL were tested for their ability to interfere with pro-thrombotic and pro-inflammatory aspects of platelet function. We are able to show that HDL significantly impaired OxLDL-induced platelet aggregation and adhesion. In gel-filtered platelets, HDL decreased both the formation of reactive oxygen species and CD40L expression. Furthermore, HDL strongly interfered with OxLDL-induced formation of platelet-neutrophil aggregates in whole blood, suggesting that platelets represent a relevant and sensitive target for HDL. The finding that HDL effectively competed with the binding of OxLDL to the platelet surface might contribute to their atheroprotective and antithrombotic properties.

Platelets directly enhance neutrophil transmigration in response to oxidised low-density lipoprotein.

Beyond their primary role in haemostasis and tissue repair, platelets are causally involved in the onset of inflammatory reactions, cell proliferation and immune response. Platelet activation and platelet binding to the endothelium result in release of chemokines and increased expression of adhesion molecules, which promote the recruitment of leukocytes that will eventually migrate across the endothelium into the tissue. Here, we provide the first evidence that platelets stimulated with oxidised low-density lipoprotein (oxLDL) directly enhance recruitment and transmigration of neutrophils, via cell-cell interaction. OxLDL immediately activates platelets, which then rapidly bind to neutrophils, foster their activation and facilitate transmigration through an endothelial monolayer. The observed effects of oxLDL on platelet-neutrophil aggregate (PNA) formation depend on incubation time, lipoprotein concentration and the degree of oxidative modification of LDL. PNA form within minutes following stimulation by oxLDL and remain for up to 1 h post stimulation, while native LDL is unable to induce platelet-neutrophil interactions. In the presence of acetylsalicylic acid the formation of PNA in response to oxLDL is virtually absent, and platelets fail to further enhance oxLDL-induced neutrophil transmigration. P2Y1 and P2Y12 inhibitors have less pronounced effects on PNA formation in response to oxLDL. Furthermore, we demonstrate that the PI3K pathway is essential for efficient neutrophil transmigration induced by oxLDL. Consequently, platelets enhance neutrophil transmigration in response to oxLDL and might thereby contribute essentially to the amplification of inflammatory processes within the vessel wall, which fosters the development of atherosclerosis.

Periodontopathogens induce expression of CD40L on human platelets via TLR2 and TLR4.

INTRODUCTION: The outstanding importance of (soluble) CD40L to cardiovascular disease (CVD) is becoming increasingly apparent as CD40L is an important mediator of thrombotic and inflammatory processes. Platelets are the main source for CD40 ligand, linking platelet stimulatory events to inflammation and adverse adaptive immune responses. Periodontitis represents a chronic dental infection by distinct gram negative bacteria that is associated with an increased risk for CVD. However, the effects of periodontopathogens on CD40L expression by platelets have not been determined. MATERIAL AND METHODS: Effects of periodontopathogens A. actinomycetemcomitans Y and P. gingivalis on the expression of CD40L were determined and the underlying receptors and pathways were investigated. 26 patients with periodontitis and 19 controls were included in the clinical part of this study. RESULTS: Periodontopathogens directly induce surface expression of CD40L in human platelets. This activation depends on plasma factors like CD14 and involves TLR2 and TLR4 but not FcγRII. Inhibition of PI3K and PLC completely abolishes bacteria-induced surface expression of CD40L. TLR2 and TLR4 agonists, for example, are also able to induce expression and release of CD40L in human platelets. In patients with periodontitis, plasma levels of soluble CD40L are elevated and positivity for P. gingivalis is associated with a statistical significant increase of soluble CD40L. CONCLUSIONS: Our data indicate an involvement of periodontopathogens in increased plasma levels of soluble CD40L in periodontitis and therefore provide a novel link between periodontitis and increased risk for CVD.

Reconstitution of PTEN activity by CK2 inhibitors and interference with the PI3-K/Akt cascade counteract the antiapoptotic effect of human stromal cells in chronic lymphocytic leukemia.

Evidence suggests that tumor microenvironment is critically involved in supporting survival of chronic lymphocytic leukemia (CLL) cells. However, the molecular mechanisms of this effect and the clinical significance are not fully understood. We applied a microenvironment model to explore the interaction between CLL cells and stromal cells and to elucidate the role of phosphatidylinositol 3 kinase (PI3-K)/Akt/phosphatase and tensin homolog detected on chromosome 10 (PTEN) cascade in this process and its in vivo relevance. Primary human stromal cells from bone marrow, lymph nodes, and spleen significantly inhibited spontaneous apoptosis of CLL cells. Pan-PI3-K inhibitors (LY294002, wortmannin, PI-103), isotype-specific inhibitors of p110α, p110ß, p110γ, and small interfering RNA against PI3-K and Akt1 counteracted the antiapoptotic effect of the stromal cells. Induction of apoptosis was associated with a decrease in phosphatidylinositol-3,4,5-triphosphate, PI3-K-p85, and dephosphorylation of phosphatidylinositol-dependent kinase-1 (PDK-1), Akt1, and PTEN. Freshly isolated peripheral blood mononuclear cells from patients with CLL (n = 44) showed significantly higher levels of phosphorylated Akt1, PDK-1, PTEN, and CK2 than healthy persons (n = 8). CK2 inhibitors (4,5,6,7-tetrabromo-1H-benzotriazole, apigenin, and 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazol) decreased phosphorylation of PTEN and Akt, induced apoptosis in CLL cells, and enhanced the response to fludarabine. In conclusion, bone marrow microenvironment modulates the PI3-K/Akt/PTEN cascade and prevents apoptosis of CLL cells. Combined inhibition of PI3-K/Akt and recovery of PTEN activity may represent a novel therapeutic concept for CLL.