Organ-specific immune response in lethal SARS-CoV-2 infection by deep spatial phenotyping.

Objectives: Immunopathology of ongoing COVID-19 global pandemic is not limited solely to pulmonary tissue, but is often associated with multi-organ complications, mechanisms of which are intensely being investigated. In this regard, the interplay between immune, stromal cells and cytokines in pulmonary and extrapulmonary infected tissues, especially in young adults (median age 46 years, range 30-53 years) without comorbidities, remains poorly characterised. Methods: We profiled lung, heart and intestinal autopsy samples from five SARS-CoV-2-infected cases for 18-20 targets to detect immune, cytokine and stromal cell status at subcellular resolution by a novel IHC-based deep-phenotyping technique, iSPOT (immunoSpatial histoPhenOmics using TSA-IHC), to assess spatial and functional patterns of immune response in situ, in lethal COVID-19 infection. Results: SARS-CoV-2-infected autopsy samples exhibit skewed counts of immune populations in all samples with organ-specific dysfunctions. Lung and ileal tissue reveal altered architecture with marked loss of tissue integrity, while lung and heart tissue show severe hyperinflammation marked by elevated TNF-α in heart tissue and additionally IL-6, IFN-γ and IL-10 cytokines in lung samples. Conclusion: With resurgence of infection in younger populations, single-cell cytokine localisation in immune and stromal structures provides important mechanistic insights into organ-specific immunopathology of naïve SARS-CoV-2 infection in the absence of other comorbidities.

Corrigendum: Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

[This corrects the article DOI: 10.3389/fmolb.2021.658932.].

Immune Response in Myocardial Injury: In Situ Hybridization and Immunohistochemistry Techniques for SARS-CoV-2 Detection in COVID-19 Autopsies.

Coronavirus disease-19 (COVID-19) is caused by the newly discovered coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While the lung remains the primary target site of COVID-19 injury, damage to myocardium, and other organs also contribute to the morbidity and mortality of this disease. There is also increasing demand to visualize viral components within tissue specimens. Here we discuss the cardiac autopsy findings of 12 intensive care unit (ICU) naïve and PCR-positive COVID-19 cases using a combination of histological, Immunohistochemical/immunofluorescent and molecular techniques. We performed SARS-CoV-2 qRT-PCR on fresh tissue from all cases; RNA-ISH and IHC for SARS-CoV-2 were performed on selected cases using FFPE tissue from heart. Eight of these patients also had positive post-mortem serology for SARS-CoV-2. Histopathologic changes in the coronary vessels and inflammation of the myocardium as well as in the endocardium were documented which support the reports of a cardiac component to the viral infection. As in the pulmonary reports, widespread platelet and fibrin thrombi were also identified in the cardiac tissue. In keeping with vaccine-induced activation of virus-specific CD4+ and CD8+ T cells, and release of cytokines such as interferon-gamma (IFNγ), we observed similar immune cellular distribution and cytokines in these patients. Immunohistochemical and immunofluorescent localisation for the viral Spike (S-protein) protein and the nucleocapsid protein (NP) were performed; presence of these aggregates may possibly contribute to cardiac ischemia and even remodelling.

Elderly Asian Patients Have Lower Revascularisation Rates and Poorer Outcomes for ST-Elevation Myocardial Infarction Compared to Younger Patients.

INTRODUCTION: There is limited information on elderly patients presenting with ST- elevation myocardial infarction (STEMI). This study aimed to study the outcomes of elderly Asian patients with STEMI compared to younger patients. MATERIALS AND METHODS: The study utilised data from 2007 to 2012 from the Singapore Myocardial Infarction Registry, a mandatory national population-based registry. Elderly patients were defined as ≥80 years of age, middle-aged to old (MAO) patients were defined as 45-80 years of age and young patients were defined as ≤45 years of age. The primary outcome of the study was 1-year mortality and secondary outcomes included in-hospital complications and mortality. RESULTS: There were 12,409 STEMI patients with 1207 (9.7%) elderly patients, 10,093 (81.3%) MAO patients and 1109 (8.9%) young patients. Elderly patients had more cardiovascular risk factors and lower rates of total percutaneous coronary intervention (26.0% vs 72.4% vs 85.5%, respectively; P <0.0001) compared to MAO and young patients. They had higher 1-year mortality (60.6% vs 18.3% vs 4.1%, respectively; P <0.0001) when compared to MAO and young patients. CONCLUSION: Elderly patients with STEMI have poorer outcomes than MAO and young patients. This is potentially attributable to a myriad of factors including age, higher burden of comorbidities and a lesser likelihood of receiving revascularisation and guideline-recommended medical therapy.

Genetic variability of RyR2 and CASQ2 genes in an Asian population.

We analyzed the coding regions of the cardiac calcium-handling genes, ryanodine receptor 2 (RyR2) and calsequestrin 2 (CASQ2) for genetic variants in a healthy Chinese population (n=95) and in a cohort of 28 sudden unexplained death victims. Mutations in RyR2 and CASQ2 have been shown to alter calcium homeostasis during excitation-contraction coupling and predispose individuals to fatal cardiac arrhythmias. The genetic screening was accomplished by denaturing high-performance liquid chromatography and DNA sequencing methods. Genetic analysis revealed the following non-synonymous genetic variations: two reported RyR2 polymorphisms; 5654G>A (G1885E) and 5656G>A (G1886S), two reported CASQ2 polymorphisms; 196A>G (T66A) and 226G>A (V76M) and one novel CASQ2 mutation; 529G>C (E177Q). The functional significance of the novel CASQ2 mutation has not been evaluated and characterized. This study shows that multiple genetic variations of the RyR2 and CASQ2 genes exist in the two study populations. The inter-individual genetic variability may underlie the different susceptibility of individuals to developing ventricular tachycardia. The research results will be valuable for which future work involving clinical and forensic samples can be based upon to distinguish potential disease-associated mutations from common polymorphisms.

Elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients.

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis; some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, IGF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TAC1. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung.

SARS-CoV virus-host interactions and comparative etiologies of acute respiratory distress syndrome as determined by transcriptional and cytokine profiling of formalin-fixed paraffin-embedded tissues.

These studies attempt to understand more fully the host response and pathogenesis associated with severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) by monitoring gene expression using formalin-fixed paraffin-embedded (FFPE) pulmonary autopsy tissues. These tissues were from patients in different hospitals in Singapore who were diagnosed with various microbial infections, including SARS-CoV, that caused acute respiratory distress syndrome (ARDS). Global expression patterns showed limited correlation between end-stage ARDS and the initiating pathogen, but when focusing on a subset of genes implicated in pulmonary pathogenesis, molecular signatures of pulmonary disease were obtained and appeared to be influenced by preexisting pulmonary complications and also bacterial components of infection. Many factors detected during pulmonary damage and repair, such as extracellular matrix (ECM) components, transforming growth factor (TGF) enhancers, acute-phase proteins, and antioxidants, were included in the molecular profiles of these ARDS lung tissues. In addition, differential expression of cytokines within these pulmonary tissues were observed, including notable genes involved in the interferon (IFN) pathway, such as Stat1, IFN regulatory factor-1 (IRF-1), interleukin-6 (IL-6), IL-8, and IL-18, that are often characterized as elevated in ARDS patients.

Proteomic analysis of mouse liver for the evaluation of effects of Scutellariae radix by liquid chromatography with tandem mass spectrometry.

Scutellariae radix or Scutellaria baicalensis is a medicinal plant that contains major flavonoids such as baicalein, baicalin, wogonin and wogonosides. The present work describes the development of an approach using proteomic analysis of mouse liver to study the effects of prolonged exposure to substances present in chemically standardized Scutellariae radix extracts. Histopathological examination of the mouse liver was compared with the proteome data. The botanical extracts were prepared using pressurized liquid extraction (PLE). A method without isotope labeling was developed, using proteolytic digestion with one- and two-dimensional liquid chromatography with tandem mass spectrometry, and was used to characterize the extent of differential protein expression in mouse liver in response to external factors such as extracts from Scutellariae radix. From the histopathological examination and proteome data, significant changes in the mouse livers were not observed for the low-dose group. The Scutellariae radix extracts at high dose were observed to cause damage at the bile duct and expression change of a number of proteins including some involved in catabolism of triglyceride-rich particles, carbohydrate metabolism, regulators of cell signaling processes, and enzymes involved in biotransformation. Thus, proteomic analysis of liver samples from mice treated with botanical extracts is a promising approach to provide information on any potential toxicity effects of the extracts. The present method also provides another means for comparing proteomes in biological samples such as liver lysates from mice subjected to different treatments.

Analysis of deaths during the severe acute respiratory syndrome (SARS) epidemic in Singapore: challenges in determining a SARS diagnosis.

CONTEXT: An outbreak of severe acute respiratory syndrome (SARS), an infectious disease attributed to a novel coronavirus, occurred in Singapore during the first quarter of 2003 and led to 204 patients with diagnosed illnesses and 26 deaths by May 2, 2003. Twenty-one percent of these patients required admission to the medical intensive care unit. During this period, the Center for Forensic Medicine, Health Sciences Authority, Singapore, performed a total of 14 postmortem examinations for probable and suspected SARS. Of these, a total of 8 were later confirmed as SARS infections. OBJECTIVE: Our series documents the difficulties encountered at autopsy during the initial phases of the SARS epidemic, when the pattern of infection and definitive diagnostic laboratory criteria were yet to be established. DESIGN: Autopsies were performed by pathologists affiliated with the Center for Forensic Medicine, Health Sciences Authority, Singapore. Tissue was accessed and read at the Tan Tock Seng Hospital, Singapore, and at the Armed Forces Institute of Pathology, Washington, DC. Autopsy tissue was submitted to the Virology Department, Singapore General Hospital, for analysis, and in situ hybridization for the SARS coronavirus was carried out at the National Institute of Infectious Diseases, Tokyo, Japan. RESULTS: Thirteen of 14 patients showed features of diffuse alveolar damage. In 8 patients, no precipitating etiology was identified, and in all of these patients, we now have laboratory confirmation of coronavirus infection. Two of the 8 patients presented at autopsy as sudden unexpected deaths, while the remaining 6 patients had been hospitalized with varying lengths of stay in the intensive care unit. In 3 patients, including the 2 sudden unexpected deaths, in situ hybridization showed the presence of virally infected cells within the lung. In 4 of the 8 SARS patients, pulmonary thromboemboli were also recognized on gross examination, while one patient had marantic cardiac valvular vegetations. CONCLUSIONS: It is unfortunate that the term atypical pneumonia has been used in conjunction with SARS. Although nonspecific by itself, the term does not accurately reflect the underlying dangers of viral pneumonia, which may progress rapidly to acute respiratory distress syndrome. We observed that the clinical spectrum of disease as seen in our autopsy series included sudden deaths. This is a worrisome finding that illustrates that viral diseases will have a spectrum of clinical presentations and that the diagnoses made for such patients must incorporate laboratory as well as clinical data.

Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore.

Severe acute respiratory syndrome (SARS) is an infectious condition caused by the SARS-associated coronavirus (SARS-CoV), a new member in the family Coronaviridae. To evaluate the lung pathology in this life-threatening respiratory illness, we studied postmortem lung sections from 8 patients who died from SARS during the spring 2003 Singapore outbreak. The predominant pattern of lung injury in all 8 cases was diffuse alveolar damage. The histology varied according to the duration of illness. Cases of 10 or fewer days' duration demonstrated acute-phase diffuse alveolar damage (DAD), airspace edema, and bronchiolar fibrin. Cases of more than 10 days' duration exhibited organizing-phase DAD, type II pneumocyte hyperplasia, squamous metaplasia, multinucleated giant cells, and acute bronchopneumonia. In acute-phase DAD, pancytokeratin staining was positive in hyaline membranes along alveolar walls and highlighted the absence of pneumocytes. Multinucleated cells were shown to be both type II pneumocytes and macrophages by pancytokeratin, thyroid transcription factor-1, and CD68 staining. SARS-CoV RNA was identified by reverse transcriptase-polymerase chain reaction in 7 of 8 cases in fresh autopsy tissue and in 8 of 8 cases in formalin-fixed, paraffin-embedded lung tissue, including the 1 negative case in fresh tissue. Understanding the pathology of DAD in SARS patients may provide the basis for therapeutic strategies. Further studies of the pathogenesis of SARS may reveal new insight into the mechanisms of DAD.