Cytokine Profiling of Plasma and Atherosclerotic Plaques in Patients Undergoing Carotid Endarterectomy.

Atherosclerotic plaques are sites of chronic inflammation with diverse cell contents and complex immune signaling. Plaque progression and destabilization are driven by the infiltration of immune cells and the cytokines that mediate their interactions. Here, we attempted to compare the systemic cytokine profiles in the blood plasma of patients with atherosclerosis and the local cytokine production, using ex vivo plaque explants from the same patients. The developed method of 41-plex xMAP data normalization allowed us to differentiate twenty-two cytokines produced by the plaque that were not readily detectable in free circulation and six cytokines elevated in blood plasma that may have other sources than atherosclerotic plaque. To verify the xMAP data on the putative atherogenesis-driving chemokines MCP-1 (CCL2), MIP-1α (CCL3), MIP-1ß (CCL4), RANTES (CCL5), and fractalkine (CX3CL1), qPCR was performed. The MIP1A (CCL3), MIP1B (CCL4), FKN (CX3CL1), and MCP1 (CCL2) genes were expressed at high levels in the plaques, whereas RANTES (CCL5) was almost absent. The expression patterns of the chemokines were restricted to the plaque cell types: the MCP1 (CCL2) gene was predominantly expressed in endothelial cells and monocytes/macrophages, MIP1A (CCL3) in monocytes/macrophages, and MIP1B (CCL4) in monocytes/macrophages and T cells. RANTES (CCL5) was restricted to T cells, while FKN (CX3CL1) was not differentially expressed. Taken together, our data indicate a plaque-specific cytokine production profile that may be a useful tool in atherosclerosis studies.

Targeted Blood Plasma Proteomics and Hemostasis Assessment of Post COVID-19 Patients with Acute Myocardial Infarction.

The molecular mechanisms underlying cardiovascular complications after the SARS-CoV-2 infection remain unknown. The goal of our study was to analyze the features of blood coagulation, platelet aggregation, and plasma proteomics in COVID-19 convalescents with AMI. The study included 66 AMI patients and 58 healthy volunteers. The groups were divided according to the anti-N IgG levels (AMI post-COVID (n = 44), AMI control (n = 22), control post-COVID (n = 31), and control (n = 27)). All participants underwent rotational thromboelastometry, thrombodynamics, impedance aggregometry, and blood plasma proteomics analysis. Both AMI groups of patients demonstrated higher values of clot growth rates, thrombus size and density, as well as the elevated levels of components of the complement system, proteins modifying the state of endothelium, acute-phase and procoagulant proteins. In comparison with AMI control, AMI post-COVID patients demonstrated decreased levels of proteins connected to inflammation and hemostasis (lipopolysaccharide-binding protein, C4b-binding protein alpha-chain, plasma protease C1 inhibitor, fibrinogen beta-chain, vitamin K-dependent protein S), and altered correlations between inflammation and fibrinolysis. A new finding is that AMI post-COVID patients opposite the AMI control group, are characterized by a less noticeable growth of acute-phase proteins and hemostatic markers that could be explained by prolonged immune system alteration after COVID-19.

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.

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.

​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.

High SARS-CoV-2 load in the nasopharynx of patients with a mild form of COVID-19 is associated with clinical deterioration regardless of the hydroxychloroquine administration.

Because of the constantly growing numbers of COVID-19 infections and deaths, attempts were undertaken to find drugs with anti-SARS-CoV-2 activity among ones already approved for other pathologies. In the framework of such attempts, in a number of in vitro, as well as in vivo, models it was shown that hydroxychloroquine (HCQ) has an effect against SARS-CoV-2. While there were not enough clinical data to support the use of HCQ, several countries including Russia have included HCQ in treatment protocols for infected patients and for prophylaxis. In the current non-randomized, observational study we evaluated the SARS-CoV-2 RNA in nasopharynx swabs from infected patients 7-10 days post symptoms with clinically mild disease and compared the viral RNA load dynamics between patients receiving HCQ (200 mg twice per day according to the Ministry of Health of Russian Federation treatment instructions, n = 33) and a control group without antiviral pharmacological therapy (n = 12). We found a statistically significant relationship between maximal RNA quantity and deterioration of patients' medical conditions, and as well we confirmed arterial hypertension to be a risk factor for people with COVID-19. However, we showed that at the dose used in the study HCQ therapy neither shortened the viral shedding period nor reduced the virus RNA load.

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.

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.