Background The novel coronavirus has a high mortality rate (over 1% for patients older than 50 years). This can only be partially ascribed to other comorbidities. A possible explanation is a factor that assures a prompt response to SARS-CoV-2 in younger people, independent from the novelty of the virus itself. A factor is believed to stimulate the immune system and provide immunity against more antigens. The only external stimulation received by healthy people is vaccination (eg, the diphtheria, tetanus, and pertussis [DTP] vaccine). One hypothesis is that vaccination helps develop specific immunity but generates sprouting immunity against antigens in transit. The underlying immunological phenomena are the “bystander effect” and “trained immunity.” The developed immunity gives protection for years until it naturally fades out. After the fifth decade of life, the immune system is almost incompetent when a viral infection occurs, and thus, at this stage, the novel coronavirus can enter the body and cause acute respiratory distress syndrome. Objective The initial aim is to demonstrate that blood monocytes and natural killer cells show overpowering hyperactivity, while CD4+ and CD8+ T cells experience impediments to their defensive functions in patients with severe SARS-CoV-2 infection. The secondary objectives are to correlate clinical data and vaccination history with laboratory immune patterns in order to identify protective factors. Subsequently, we are also interested in characterizing the phenotypes and state of the degree of activation of peripheral blood mononuclear cells, including monocytes, natural killer cells, and CD4+ and CD8+ T cells, in healthy subjects vaccinated with the Pfizer vaccine. Methods Data will be collected using the following 3 approaches: (1) an experimental analysis to study the innate immune response and to identify genetic profiles; (2) an epidemiological analysis to identify the patients’ vaccination history; and (3) a clinical analysis to detect the immunological profile. Results The protocol was approved by the Ethics Committee on April 16, 2020, and the study started on April 27, 2020. As of February 2021, enrollment has been completed. Immunological analysis is ongoing, and we expect to complete this analysis by December 2022. Conclusions We will recognize different populations of patients, each one with a specific immunological pattern in terms of cytokines, soluble factor serum levels, and immune cell activity. Anamnestic data, such as preceding vaccinations and comorbidities, biochemical findings like lymphocyte immunophenotyping, and pre-existing persistent cytomegalovirus infection, allow depicting the risk profile of severe COVID-19. Proof of the roles of these immunological phenomena in the development of COVID-19 can be the basis for the implementation of therapeutic immunomodulatory treatments. Trial Registration ClinicalTrials.gov NCT04375176; https://clinicaltrials.gov/ct2/show/NCT04375176 International Registered Report Identifier (IRRID) DERR1-10.2196/29892

UNSTRUCTURED The novel coronavirus has a high mortality rate (over 1% for patients older than 50). This could be only partially ascribed to other comorbidities. Possible explanation could be something which assures the ability to prompt response to SARS-CoV-2 in younger people independently from the novelty of the virus itself. Something stimulated the immune system and it scattered immunity against more antigens. The only external stimulation, which healthy people receive, is vaccination (i.e. diphtheria, tetanus, and pertussis (DTP) vaccine). One hypothesis is that vaccination develops the specific immunity but generates a sprouting immunity against antigens in transit. The underlying immunological phenomena are “bystander effect” and “trained immunity”. The developed immunity gives protection for years until the natural fade out. After the fifth decade a viral infection will find immune system almost incompetent and the novel coronavirus bursts into the body, developing an ARDS. The first study’s aim is to demonstrate that monocytes, NK, CD4 + and CD8 + T cells, in patients with severe infection to SARS-CoV-2, show an overpowering hyperactivity. The secondary objectives are to correlate clinical data and vaccination history with laboratory immune pattern, to identify protective factors. Four categories of 30 patients will be analyzed: A) Asymptomatic patients; B) Mildly symptomatic patients: with fever, tiredness, dry cough, diarrhea, ect; C) Patients with diagnosis of pneumonia with “low risk” score; D) Patients with diagnosis of pneumonia with “moderate/high” risk score. Data will be collected using 3 approaches: An experimental analysis to study the innate immune response and to identify the genetic profiles; An epidemiological analysis to identify the patients’ vaccination history; A clinical analysis to detect the immunological profile. We suppose to recognize different populations of patients, each one with a specific immunological pattern in terms of cytokines, soluble factors serum level and immune cells activity. Anamnestic data such as preceding vaccinations and comorbidities, biochemical data findings as lymphocyte immunophenotyping and pre-existing persistent cytomegalovirus infection allow depicting the risk profile of severe COVID-19. The proof of a role of these immunological phenomena on the development of COVID-19 are bases for implementation of therapeutic immunomodulatory treatments.

Three-dimensional (3D) chromatin organization has a key role in defining the transcription program of cells during development. Its alteration is the cause of gene expression changes responsible for several diseases. Thus, we need new tools to study this aspect of gene expression regulation. To this end, ChromEM was recently developed: this is an electron-microscopy staining technique that selectively marks nuclear DNA without altering its structure and, thus, allows better visualization of 3D chromatin conformation. However, despite increasingly frequent application of this staining technique on cells, it has not yet been applied to visualize chromatin ultrastructure in tissues. Here, we provide a protocol to carry out ChromEM on myocardial tissue harvested from the left ventricles of C57BL/6J mice and use this in combination with transmission electron microscopy (TEM) to measure some morphological parameters of peripheral heterochromatin in cardiomyocytes. This protocol could also be used, in combination with electron tomography, to study 3D chromatin organization in cardiomyocytes in different aspects of heart pathobiology (e.g., heart development, cardiac aging, and heart failure) as well as help to set-up ChromEM in other tissues.