Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)-Specific T Cells and Antibodies in Coronavirus Disease 2019 (COVID-19) Protection: A Prospective Study.

BACKGROUND: During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection. METHODS: In 5340 Moscow residents, we evaluated anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFN-γ and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan-Meier estimator method, for up to 300 days postinclusion. RESULTS: We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection. CONCLUSIONS: We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti-COVID-19 policies. Clinical Trials Registration. NCT04898140.

Comprehensive Cytokine Profiling of Patients with COVID-19 Receiving Tocilizumab Therapy.

Coronavirus disease 2019 (COVID-19) is characterized by immune activation in response to viral spread, in severe cases leading to the development of cytokine storm syndrome (CSS) and increased mortality. Despite its importance in prognosis, the pathophysiological mechanisms of CSS in COVID-19 remain to be defined. Towards this goal, we analyzed cytokine profiles and their interrelation in regard to anti-cytokine treatment with tocilizumab in 98 hospitalized patients with COVID-19. We performed a multiplex measurement of 41 circulating cytokines in the plasma of patients on admission and 3-5 days after, during the follow-up. Then we analyzed the patient groups separated in two ways: according to the clusterization of their blood cytokines and based on the administration of tocilizumab therapy. Patients with and without CSS formed distinct clusters according to their cytokine concentration changes. However, the tocilizumab therapy, administered based on the standard clinical and laboratory criteria, did not fully correspond to those clusters of CSS. Furthermore, among all cytokines, IL-6, IL-1RA, IL-10, and G-CSF demonstrated the most prominent differences between patients with and without clinical endpoints, while only IL-1RA was prognostically significant in both groups of patients with and without tocilizumab therapy, decreasing in the former and increasing in the latter during the follow-up period. Thus, CSS in COVID-19, characterized by a correlated release of multiple cytokines, does not fully correspond to the standard parameters of disease severity. Analysis of the cytokine signature, including the IL-1RA level in addition to standard clinical and laboratory parameters may be useful to define the onset of a cytokine storm in COVID-19 as well as the indications for anti-cytokine therapy.

Flow analysis of individual blood extracellular vesicles in acute coronary syndrome.

A diverse population of small extracellular vesicles (EVs) that are released by various cells has been characterized predominantly in bulk, a procedure whereby the individual characteristics of EVs are lost. Here, we used a new nanotechnology-based flow cytometric analysis to characterize the antigenic composition of individual EVs in patients with acute coronary syndrome (ACS). Plasma EVs were captured with 15-nm magnetic nanoparticles coupled to antibodies against CD31 (predominantly an endothelial marker), CD41a (a marker for platelets), and CD63 or MHC class I (common EV markers). The total amounts of EVs were higher in the ACS patients than in the controls, predominantly due to the contribution of patients with acute myocardial infarction. For all captured fractions, the differences in the EV amounts were restricted to CD41a+ EVs. The increase in the numbers of EVs in the ACS patients, predominantly of platelet origin, probably reflects platelet activation and may indicate disease progression.

Activation of T lymphocytes in atherosclerotic plaques.

OBJECTIVE: To decipher the immunologic mechanisms of plaque maturation and rupture, it is necessary to analyze the phenotypes and distribution of individual lymphocytes that migrate to the plaques, as well as their activation at different stages of plaque formation. METHODS AND RESULTS: We developed a protocol to isolate plaque-residing immune cells and analyze their status using polychromatic flow cytometry. We found that the composition and phenotype of T lymphocytes in the plaques differs from that in blood. CD4 and, in particular, CD8(+) T cells in plaques are highly activated; the fraction of CD8 T cells coexpressing CD25 and human leukocyte antigen-D related in plaques was 6 times as large as in blood. CONCLUSIONS: The first flow-cytoanalysis of individual T cells in atherosclerotic plaques indicates that plaques represent a separate immunologic compartment from blood with lymphocytes characterized by a high level of T-cell activation, which is compatible with the presence of antigen(s) that trigger infiltration activation of these cells. The ability to isolate and characterize these cells may lead to the identification of such antigens.

​Circulating Cytokines in Myocardial Infarction Are Associated With Coronary Blood Flow.

BACKGROUND: The level of systemic inflammation correlates with the severity of the clinical course of acute myocardial infarction (AMI). It has been shown that circulating cytokines and endothelial dysfunction play an important role in the process of clot formation. The aim of our study was to assess the concentration of various circulating cytokines, endothelial function and blood clotting in AMI patients depending on the blood flow through the infarction-related artery (IRA). METHODS: We included 75 patients with AMI. 58 presented with ST-elevation myocardial infarction (STEMI) and 17 had non-ST-elevation myocardial infarction (non-STEMI). A flow-mediated dilation test (FMD test), thrombodynamics and rotational thromboelastometry as well as assessment of 14 serum cytokines using xMAP technology were performed. FINDINGS: Non-STEMI-patients were characterized by higher levels of MDC, MIP-1ß, TNF-α. Moreover, we observed that patients with impaired blood flow through the IRA (TIMI flow 0-1) had higher average and initial clot growth rates, earlier onset of spontaneous clots, C-reactive protein (CRP) and IL-10 compared to patients with preserved blood flow through the IRA (TIMI flow 2-3). Patients with TIMI 2-3 blood flow had higher level of IP-10. IL-10 correlated with CRP and pro-inflammatory cytokines levels, initial clot growth rate and clot lysis time in TIMI 0-1 patients. All these differences were statistically significant. INTERPRETATION: We demonstrated that concentrations of the inflammatory cytokines correlate not only with the form of myocardial infarction (STEMI or non-STEMI), but also with the blood flow through the infarct-related artery. Inflammatory response, functional state of endothelium, and clot formation are closely linked with each other. A combination of these parameters affects the patency of the infarct-related artery.

Long-term Success of Cardioneuroablation in a Patient with Tachycardia-bradycardia Syndrome and Syncope.

A case of successful treatment for syncopal episodes caused by intermittent atrioventricular block in a patient with paroxysmal atrial fibrillation/atrial flutter using cardioneuroablation is presented.

Differential clusterization of soluble and extracellular vesicle-associated cytokines in myocardial infarction.

A proinflammatory dysregulation of cytokine release is associated with various diseases, in particular with those of infectious etiology, as well as with cardiovascular diseases (CVD). We showed earlier that cytokines are released in two forms, soluble and in association with extracellular vesicles (EVs). Here, we investigated the patterns of expression and clustering of soluble and EV-associated cytokines in patients with ST-elevation myocardial infarction (STEMI). We collected plasma samples from 48 volunteers without CVD and 62 patients with STEMI, separated soluble and EV fractions, and analyzed them for 33 cytokines using a multiplexed bead-based assay. We identified soluble and EV-associated cytokines that are upregulated in STEMI and form correlative clusters. Several clustered soluble cytokines were expressed almost exclusively in patients with STEMI. EV-associated cytokines were largely not affected by STEMI, except for pro-inflammatory cytokines IL-6, IL-18, and MIG, as well as anti-inflammatory IL-2 that were upregulated in a correlated fashion. Our results demonstrated that soluble cytokines in patients with STEMI are upregulated in a coordinated fashion in contrast to the mainly unaffected system of EV-associated cytokines. Identification of cytokine clusters affected differently by STEMI now permits investigation of their differential contributions to this pathology.

Productive Cytomegalovirus Infection Is Associated With Impaired Endothelial Function in ST-Elevation Myocardial Infarction.

BACKGROUND: An association between productive cytomegalovirus infection and atherosclerosis was shown recently in several trials, including a previous study of ours. However, the mechanism involved in this association is still under investigation. Here, we addressed the interaction between productive cytomegalovirus infection and endothelial function in patients with ST-elevation myocardial infarction (STEMI). METHODS: We analyzed the presence of cytomegaloviral DNA in plasma and endothelial function in 33 patients with STEMI and 33 volunteers without cardiovascular diseases, using real-time polymerase chain reaction (PCR) and a noninvasive test of flow-mediated dilation. RESULTS: Both the frequency of presence and the load of cytomegaloviral DNA were higher in plasma of patients with STEMI than those in controls. This difference was independent of other cardiovascular risk factors (7.38 [1.36-40.07]; P = 0.02). The results of the flow-mediated dilation test were lower in patients in STEMI than in controls (5.0% [2.65%-3.09%] vs 12. %5 [7.5%-15.15%]; P = 0.004) and correlated negatively with the cytomegaloviral DNA load (Spearman R = -0.407; P = 0.019) independently of other cardiovascular risk factors. CONCLUSIONS: Productive cytomegalovirus infection in patients with STEMI correlated negatively with endothelial function independently of other cardiovascular risk factors. The impact of cytomegalovirus on endothelial function may explain the role of cytomegalovirus in cardiovascular prognosis.

Dynamics of coagulopathy in patients with different COVID-19 severity.

With the progress of COVID-19 studies, it became evident that SARS-CoV-2 infection is often associated with thrombotic complications. The goal of our present study was to evaluate which component of clot formation process including endothelial function, platelets aggregation and plasma coagulation, as well as endogenous fibrinolysis in patients with COVID-19 correlates with the severity of the disease. We prospectively included 58 patients with COVID-19 and 47 healthy volunteers as a control group that we recruited before the pandemic started. It turns out that plasma coagulation with subsequent platelet aggregation, but not endothelial function, correlates with the severity of the COVID-19. IL-6 blockade may play a beneficial role in COVID-19 induced coagulopathy.

Monocytes of Different Subsets in Complexes with Platelets in Patients with Myocardial Infarction.

Acute myocardial infarction (AMI) is associated with activation of various cells, including platelets that form monocyte-platelet complexes (MPCs). Here, we analysed MPC in vivo and in vitro and investigated the abilities of different monocyte subclasses to form MPC, the characteristics of the cells involved in MPC formation and MPC changes in AMI. We identified MPC by co-staining for platelet antigen CD41a and monocyte antigens CD14 and CD16. Platelet activation was evaluated from expression of phosphatidylserine as revealed by annexin V. Our results confirm published data and provide new information regarding the patterns of MPC in AMI patients. We found that the patterns of platelet aggregation with monocytes were different in AMI patients and controls: (1) in AMI patients, MPC formed by intermediate monocytes carry more platelets whereas in healthy controls more platelets aggregated with classical monocytes; (2) the numbers of MPC in AMI patients, being already higher than in controls, were further increased if these patients suffered various in-hospital complications; (3) on the basis of the CD41a fluorescence of the antibody-stained MPC, some of the aggregates seem to consist of monocytes and platelet-derived extracellular vesicles (EVs); (4) aggregation of monocytes with platelet EV occurred in in vitro experiments; and (5) these experiments demonstrated that monocytes from AMI patients aggregate with both platelets and platelet EVs more efficiently than do monocytes from controls. MPC in AMI patients may play an important role in this pathology.

Ex vivo culture of human atherosclerotic plaques: A model to study immune cells in atherogenesis.

BACKGROUND AND AIMS: The mechanisms that drive atherosclerotic plaque progression and destabilization in humans remain largely unknown. Laboratory models are needed to study these mechanisms under controlled conditions. The aim of this study was to establish a new ex vivo model of human atherosclerotic plaques that preserves the main cell types in plaques and the extracellular components in the context of native cytoarchitecture. METHODS: Atherosclerotic plaques from carotid arteries of 28 patients undergoing carotid endarterectomy were dissected and cultured. At various time-points, samples were collected and analysed histologically. After enzymatic digestion, single cells were analysed with flow cytometry. Moreover, tissue cytokine production was evaluated. RESULTS: We optimised the plaque dissection protocol by cutting plaques into circular segments that we cultured on collagen rafts at the medium-air interface, thus keeping them well oxygenated. With this technique, the relative presence of T and B lymphocytes did not change significantly during culture, and the sizes of lymphocyte subsets remained stable after day 4 of culture. Macrophages, smooth muscle cells, and fibroblasts with collagen fibres, as well as T and B lymphocyte subsets and CD16 natural killer cells, remained largely preserved for 19 days of culture, with a continuous production of inflammatory cytokines and chemokines. CONCLUSIONS: Our new model of ex vivo human atherosclerotic plaques, which preserves the main subsets of immune cells in the context of tissue cytoarchitecture, may be used to investigate important aspects of atherogenesis, in particular, the functions of immune cells under controlled laboratory conditions.

Cytomegalovirus-Productive Infection Is Associated With Acute Coronary Syndrome.

BACKGROUND: Although an association between human herpesvirus (HHV) infection and atherosclerosis has been suggested, the data supporting such an association are controversial and, in most cases, are based on serological evidence or on the presence of cell-associated HHV DNA, which do not report about actual viral replication. We quantified the DNA of all 8 types of HHVs in plasma, in which their presence is evidence of viral replication. METHODS AND RESULTS: Using quantitative real-time polymerase chain reaction, we evaluated the presence of HHV DNA in blood samples obtained at the time of hospitalization from 71 patients with acute coronary syndrome, 26 patients with stable coronary artery disease, and 53 healthy volunteers and in atherosclerotic plaques of 22 patients with peripheral artery disease who underwent endarterectomy. HHV-5 (cytomegalovirus [CMV]) was the only HHV with a level that was higher in acute coronary syndrome patients than in the control group and that correlated with the level of high-sensitivity C-reactive protein. The numbers of effector memory T cells positively correlated with the numbers of CMV genome copies in carotid arteries plaques, whereas the numbers of central memory T cells negatively correlated with CMV copy numbers. CONCLUSIONS: Of all HHV levels, only CMV was higher in patients with stable coronary artery disease and acute coronary syndrome than in the healthy group, and its load correlated with the level of high-sensitivity C-reactive protein. The level of CMV in atherosclerotic plaques correlated with the state of immunoactivation of lymphocytes in plaques, suggesting that the reactivation of CMV may contribute to the immune activation associated with the progression of atherosclerosis.

Remote ischemic preconditioning and endothelial function in patients with acute myocardial infarction and primary PCI.

BACKGROUND: Remote ischemic preconditioning by transient limb ischemia reduces myocardial ischemia-reperfusion injury in patients undergoing percutaneous coronary intervention. The aim of the study we report here was to assess the effect of remote ischemic preconditioning on endothelial function in patients with acute myocardial infarction who underwent primary percutaneous coronary intervention. METHODS: Forty-eight patients with acute myocardial infarction were enrolled. All participants were randomly divided into 2 groups. In Group I (n = 23), remote ischemic preconditioning was performed before primary percutaneous coronary intervention (intermittent arm ischemia-reperfusion through 4 cycles of 5-minute inflation and 5-minute deflation of a blood-pressure cuff to 200 mm Hg). In Group II (n = 25), standard percutaneous coronary intervention without preconditioning was performed. We assessed endothelial function using the flow-mediated dilation test on baseline, then within 1-3 hours after percutaneous coronary intervention, and again on days 2 and 7 after percutaneous coronary intervention. RESULTS: The brachial artery flow-mediated dilation results were significantly higher on the first day after primary percutaneous coronary intervention in the preconditioning group (Group I) than in the control group (Group II) (12.1% vs 0.0%, P = .03, and 11.1% vs 6.3%, P = .016, respectively), and this difference remained on the seventh day (12.3% vs 7.4%, P = .0005, respectively). CONCLUSION: We demonstrated for the first time that remote ischemic preconditioning before primary percutaneous coronary intervention significantly improves endothelial function in patients with acute myocardial infarction, and this effect remains constant for at least a week. We suppose that the improvement of endothelial function may be one of the possible explanations of the effect of remote ischemic preconditioning.

Brachial artery flow-mediated dilation in patients with Tako-tsubo cardiomyopathy.

BACKGROUND: Tako-Tsubo cardiomyopathy (TTC) is a heart syndrome associated with transient myocardial contractile dysfunction. The pathogenesis of TTC remains unclear. The purpose of this study was to investigate brachial artery flow-mediated dilation (FMD) in patients with TTC. METHODS: The results of FMD tests of 4 women with TTC were compared with the results from 18 women with ST-elevation acute myocardial infarction (STEMI) and from 26 healthy female volunteers. FMD tests in all patients were performed within 24 hours of admission and again at 1-3 weeks as a follow-up. RESULTS: The FMD levels were significantly lower at the acute phase in patients with TTC than in patients with STEMI and in healthy volunteers (P <.01). After 1-3 weeks, the FMD test results of patients with TTC had greatly increased, and no significant differences were found between these results and the results from patients with STEMI (P >.05). Also, there were no significant differences in the FMD test results between the group of patients with TTC and the group of healthy volunteers (P >.05). CONCLUSIONS: There is a pronounced and reversible endothelial dysfunction in patients with TTC, which can impair myocardial perfusion.

Cardiogenic shock: the role of inflammation.

Cardiogenic shock (CS) is the leading cause of death in patients with acute myocardial infarction (MI) and we badly need new approaches in its treatment. It has been demonstrated that a number of inflammatory cytokines (IL-1ß, IL-6, IL-8, TNF-α, CRP, soluble adhesion molecules, complement system etc) are elevated in acute MI complicated by CS. Baseline levels of pro- inflammatory cytokines have predictive value for the development of CS and subsequent mortality. The deleterious effects of pro- inflammatory cytokines may be due to excessive nitric oxide production by enzyme named NOS. However in multicenter randomized TRIUMPH study non-selective NOS inhibition was ineffective in the treatment of cardiogenic shock. A challenging subject of future studies will be treatment of CS with specific inhibitors of inducible isoform of NOS. Considering the results of treatment of patients with septic shock it would be reasonable to study the effects of small doses of corticosteroids and hemofiltration in patients with CS and signs of SIRS.

Total occlusion of the infarct-related coronary artery correlates with brachial artery flow-mediated dilation in patients with ST-elevation myocardial infarction.

BACKGROUND: Occlusion of the coronary artery is the main cause of ST-elevation myocardial infarction (STEMI). In some patients, it is followed by early spontaneous thrombolysis. In this study, we tried to determine whether spontaneous thrombolysis in the infarct-related artery (IRA) correlates with the state of the endothelium, which we assessed using the brachial artery flow-mediated dilation (FMD) test. METHODS: 52 patients with STEMI were included in the study. Based on the results of coronary angiography performed during the first three days of STEMI, the patients were divided into two groups: Group I (n=33), consisting of patients with remaining total occlusion of the IRA, and Group II (n=19), consisting of patients with spontaneous thrombolysis. We assessed the endothelial function, using the brachial artery FMD test. RESULTS: In Group I, during the first three days of STEMI, brachial artery FMD results were significantly lower than those in Group II: 5.41+/- (3.23-7.41)% versus 10.81+/-(8.00-14.89)%, respectively; P=0.000036. After 7-14 days, this difference disappeared because of significant elevation of the FMD levels in the first group. Group I was characterized by higher levels of high sensitive C-reactive protein (CRP), cholesterol, and cholesterol-LDL and lower usage of angiotensin-converting enzyme (ACE) inhibitors before STEMI. CONCLUSION: The data presented above reveal that spontaneous coronary thrombolysis in patients with acute STEMI is associated with a preserved endothelium-dependent vasodilator response in the brachial artery. It can depend on the levels of hs-CRP, of fasting glucose, and of ACE-inhibitors from previous treatment.

The antiplatelet effect of atorvastatin in patients with acute coronary syndrome depends on the hs-CRP level.

BACKGROUND: In data we published earlier, there is a correlation between platelet aggregation in patients with acute coronary syndrome (ACS) who are receiving aspirin and elevated hsCRP-level. We suggested that antiplatelet action of statins, which are known to lower hsCRP-levels, could be especially pronounced in patients with high levels of hsCRP. METHODS AND RESULTS: 54 patients with ACS without ST-segment elevation were included in this study. All patients received aspirin 160-325 mg daily. In addition to aspirin, some patients received atorvastatin 40-80 mg/d (n=19) or 300 mg of clopidogrel followed by 75 mg/d (n=15). HsCRP-levels and ADP-induced platelet aggregation were assessed on the first and on the eight days of treatment. Patients were divided into subgroups according to initial hsCRP-levels and treatment. In atorvastatin/high-CRP subgroup, the level of aggregation was about three times lower after eight days than it was on the first day. In contrast, in atorvastatin/low-CRP subgroup the level of platelet aggregation did not change during the same period. The effect of clopidogrel did not depend on hsCRP-level. In control group (patients treated with aspirin alone), platelet aggregation did not change with time. CONCLUSION: There is a correlation between antiplatelet effect of atorvastatin and initial hsCRP-level. The antiplatelet effect of clopidogrel does not depend on hsCRP-level.