In Vivo Mapping of Myocardial Injury Outside the Infarct Zone: Tissue at an Intermediate Pathological State.

BACKGROUND: The goal was to determine the feasibility of mapping the injured-but-not-infarcted myocardium using 99mTc-duramycin in the postischemic heart, with spatial information for its characterization as a pathophysiologically intermediate tissue, which is neither normal nor infarcted. METHODS AND RESULTS: Coronary occlusion was conducted in Sprague Dawley rats with preconditioning and 30-minute ligation. In vivo single-photon emission computed tomography was acquired after 3 hours (n=6) using 99mTc-duramycin, a phosphatidylethanolamine-specific radiopharmaceutical. The 99mTc-duramycin+ areas were compared with infarct and area-at-risk (n=8). Cardiomyocytes and endothelial cells were isolated for gene expression profiling. Cardiac function was measured with echocardiography (n=6) at 4 weeks. In vivo imaging with 99mTc-duramycin identified the infarct (3.9±2.4% of the left ventricle and an extensive area 23.7±2.2% of the left ventricle) with diffuse signal outside the infarct, which is pathologically between normal and infarcted (apoptosis 1.8±1.6, 8.9±4.2, 13.6±3.8%; VCAM-1 [vascular cell adhesion molecule 1] 3.2±0.8, 9.8±4.1, 15.9±4.2/mm2; tyrosine hydroxylase 14.9±2.8, 8.6±4.4, 5.6±2.2/mm2), with heterogeneous changes including scattered micronecrosis, wavy myofibrils, hydropic change, and glycogen accumulation. The 99mTc-duramycin+ tissue is quantitatively smaller than the area-at-risk (26.7% versus 34.4% of the left ventricle, P=0.008). Compared with infarct, gene expression in the 99mTc-duramycin+-noninfarct tissue indicated a greater prosurvival ratio (BCL2/BAX [B-cell lymphoma 2/BCL2-associated X] 7.8 versus 5.7 [cardiomyocytes], 3.7 versus 3.2 [endothelial]), and an upregulation of ion channels in electrophysiology. There was decreased contractility at 4 weeks (regional fractional shortening -8.6%, P<0.05; circumferential strain -52.9%, P<0.05). CONCLUSIONS: The injured-but-not-infarcted tissue, being an intermediate zone between normal and infarct, is mapped in vivo using phosphatidylethanolamine-based imaging. The intermediate zone contributes significantly to cardiac dysfunction.

Prolonged exposure to lung-derived cytokines is associated with activation of microglia in patients with COVID-19.

BACKGROUNDSurvivors of pneumonia, including SARS-CoV-2 pneumonia, are at increased risk for cognitive dysfunction and dementia. In rodent models, cognitive dysfunction following pneumonia has been linked to the systemic release of lung-derived pro-inflammatory cytokines. Microglia are poised to respond to inflammatory signals from the circulation, and their dysfunction has been linked to cognitive impairment in murine models of dementia and in humans.METHODSWe measured levels of 55 cytokines and chemokines in bronchoalveolar lavage fluid and plasma from 341 patients with respiratory failure and 13 healthy controls, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. We used flow cytometry to sort neuroimmune cells from postmortem brain tissue from 5 patients who died from COVID-19 and 3 patients who died from other causes for single-cell RNA-sequencing.RESULTSMicroglia from patients with COVID-19 exhibited a transcriptomic signature suggestive of their activation by circulating pro-inflammatory cytokines. Peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, but cumulative cytokine exposure was higher in patients with COVID-19. Treatment with corticosteroids reduced expression of COVID-19-specific cytokines.CONCLUSIONProlonged lung inflammation results in sustained elevations in circulating cytokines in patients with SARS-CoV-2 pneumonia compared with those with pneumonia secondary to other pathogens. Microglia from patients with COVID-19 exhibit transcriptional responses to inflammatory cytokines. These findings support data from rodent models causally linking systemic inflammation with cognitive dysfunction in pneumonia and support further investigation into the role of microglia in pneumonia-related cognitive dysfunction.FUNDINGSCRIPT U19AI135964, UL1TR001422, P01AG049665, P01HL154998, R01HL149883, R01LM013337, R01HL153122, R01HL147290, R01HL147575, R01HL158139, R01ES034350, R01ES027574, I01CX001777, U01TR003528, R21AG075423, T32AG020506, F31AG071225, T32HL076139.

Consensus statement on the processing, interpretation and reporting of temporal artery biopsy for arteritis.

Giant cell arteritis (GCA) is the most common systemic vasculitis in adults in Europe and North America, typically involving the extra-cranial branches of the carotid arteries and the thoracic aorta. Despite advances in noninvasive imaging, temporal artery biopsy (TAB) remains the gold standard for establishing a GCA diagnosis. The processing of TAB depends largely on individual institutional protocol, and the interpretation and reporting practices vary among pathologists. To address this lack of uniformity, the Society for Cardiovascular Pathology formed a committee tasked with establishing consensus guidelines for the processing, interpretation, and reporting of TAB specimens, based on the existing literature. This consensus statement includes a discussion of the differential diagnoses including other forms of arteritis and noninflammatory changes of the temporal artery.

Don't judge a book by its cover: a case report of apolipoprotein A-IV cardiac amyloidosis.

Background: To date, at least 20 different amyloidogenic proteins have been documented. Growing evidence suggests that despite being part of the universal amyloid proteome, apolipoprotein A-IV can be amyloidogenic, accounting for less than 1% of cases. Case summary: A 75-year-old woman was admitted for paroxysmal nocturnal dyspnoea and intermittent exertional shortness of breath and was found to be in acute heart failure. The patient underwent intravenous diuretic therapy and was discharged after decongestion. She then underwent a battery of outpatient tests to determine aetiology of her heart failure. Cardiac magnetic resonance imaging showed severe concentric left ventricular hypertrophy and diffuse late gadolinium enhancement, concerning for amyloidosis, but serologic evaluation for amyloidogenic light chain (AL) amyloidosis was negative. Tc 99m pyrophosphate (PYP) scan showed Grade 2 uptake at 1 h that was only moderately suggestive of transthyretin (TTR) amyloidosis. She ultimately received a right heart catheterization and endomyocardial biopsy, which showed apolipoprotein A-IV amyloid deposition within Congo red-positive areas of the endomyocardial specimen. The patient continues to report dyspnoea on exertion but has avoided additional heart failure admissions with intensification of her diuretic regimen. Discussion: In this case, nuclear PYP scan to evaluate for TTR amyloidosis demonstrated focal PYP uptake, but endomyocardial biopsy demonstrated apolipoprotein A-IV deposition without evidence of TTR amyloidosis. Our case increases knowledge of this rare form of amyloidosis, suggests that it may result in false positive nuclear PYP results, and highlights the importance of its evaluation, particularly in circumstances in which investigations do not reveal definitive evidence of AL or TTR amyloidosis.

Prolonged exposure to lung-derived cytokines is associated with inflammatory activation of microglia in patients with COVID-19.

Neurological impairment is the most common finding in patients with post-acute sequelae of COVID-19. Furthermore, survivors of pneumonia from any cause have an elevated risk of dementia1-4. Dysfunction in microglia, the primary immune cell in the brain, has been linked to cognitive impairment in murine models of dementia and in humans5. Here, we report a transcriptional response in human microglia collected from patients who died following COVID-19 suggestive of their activation by TNF-α and other circulating pro-inflammatory cytokines. Consistent with these findings, the levels of 55 alveolar and plasma cytokines were elevated in a cohort of 341 patients with respiratory failure, including 93 unvaccinated patients with COVID-19 and 203 patients with other causes of pneumonia. While peak levels of pro-inflammatory cytokines were similar in patients with pneumonia irrespective of etiology, cumulative cytokine exposure was higher in patients with COVID-19. Corticosteroid treatment, which has been shown to be beneficial in patients with COVID-196, was associated with lower levels of CXCL10, CCL8, and CCL2-molecules that sustain inflammatory circuits between alveolar macrophages harboring SARS-CoV-2 and activated T cells7. These findings suggest that corticosteroids may break this cycle and decrease systemic exposure to lung-derived cytokines and inflammatory activation of microglia in patients with COVID-19.

Single-cell profiling reveals inflammatory polarization of human carotid versus femoral plaque leukocytes.

Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 patients undergoing femoral (n = 23) or carotid (n = 26) endarterectomy using single-cell RNA-Seq (scRNA-Seq; n = 13), flow cytometry (n = 24), and IHC (n = 12). Comparative scRNA-Seq of CD45+-selected leukocytes from femoral (n = 9; 35,265 cells) and carotid (n = 4; 30,655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised higher proportions of myeloid cells in carotid plaques, whereas noninflammatory foam cell-like macrophages and LYVE1-overexpressing macrophages comprised higher proportions of myeloid cells in femoral plaque (P < 0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively antiinflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. In conclusion, human femoral plaques exhibit distinct macrophage phenotypic and transcriptional profiles as well as diminished CD8+ T cell populations compared with human carotid plaques.

Liver Pathology and SARS-CoV-2 Detection in Formalin-Fixed Tissue of Patients With COVID-19.

OBJECTIVES: The novel coronavirus, severe acute respiratory syndrome coronavirus 2, causing coronavirus disease 2019 (COVID-19) remains a global health threat and a significant source of human morbidity and mortality. While the virus primarily induces lung injury, it also has been reported to cause hepatic sequelae. METHODS: We aimed to detect the virus in formalin-fixed tissue blocks and document the liver injury patterns in patients with COVID-19 compared with a control group. RESULTS: We were able to detect viral RNA in the bronchioalveolar cell blocks (12/12, 100%) and formalin-fixed, paraffin-embedded tissue of the lung (8/8, 100%) and liver (4/9, 44%) of patients with COVID-19. Although the peak values of the main liver enzymes and bilirubin were higher in the patients with COVID-19 compared with the control group, the differences were not significant. The main histologic findings were minimal to focal mild portal tract chronic inflammation (7/8, 88%, P < .05) and mild focal lobular activity (6/8, 75%, P = .06). CONCLUSIONS: We found that most patients who died of COVID-19 had evidence of mild focal hepatitis clinically and histologically; however, the virus was detected in less than half of the cases.

Bone marrow-derived AXL tyrosine kinase promotes mitogenic crosstalk and cardiac allograft vasculopathy.

Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.

A Postmortem Portrait of the Coronavirus Disease 2019 (COVID-19) Pandemic: A Large Multi-institutional Autopsy Survey Study.

CONTEXT.­: This study represents the largest compilation to date of clinical and postmortem data from decedents with coronavirus disease 2019 (COVID-19). It will augment previously published small series of autopsy case reports, refine clinicopathologic considerations, and improve the accuracy of future vital statistical reporting. OBJECTIVE.­: To accurately reflect the preexisting diseases and pathologic conditions of decedents with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) infection through autopsy. DESIGN.­: Comprehensive data from 135 autopsy evaluations of COVID-19-positive decedents is presented, including histologic assessment. Postmortem examinations were performed by 36 pathologists at 19 medical centers or forensic institutions in the United States and Brazil. Data from each autopsy were collected through the online submission of multiple-choice and open-ended survey responses. RESULTS.­: Patients dying of or with COVID-19 had an average of 8.89 pathologic conditions documented at autopsy, spanning a combination of prior chronic disease and acute conditions acquired during hospitalization. Virtually all decedents were cited as having more than 1 preexisting condition, encompassing an average of 2.88 such diseases each. Clinical conditions during terminal hospitalization were cited 395 times for the 135 autopsied decedents and predominantly encompassed acute failure of multiple organ systems and/or impaired coagulation. Myocarditis was rarely cited. CONCLUSIONS.­: Cause-of-death statements in both autopsy reports and death certificates may not encompass the severity or spectrum of comorbid conditions in those dying of or with COVID-19. If supported by additional research, this finding may have implications for public health decisions and reporting moving forward through the pandemic.

Circuits between infected macrophages and T cells in SARS-CoV-2 pneumonia.

Some patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) develop severe pneumonia and acute respiratory distress syndrome1 (ARDS). Distinct clinical features in these patients have led to speculation that the immune response to virus in the SARS-CoV-2-infected alveolus differs from that in other types of pneumonia2. Here we investigate SARS-CoV-2 pathobiology by characterizing the immune response in the alveoli of patients infected with the virus. We collected bronchoalveolar lavage fluid samples from 88 patients with SARS-CoV-2-induced respiratory failure and 211 patients with known or suspected pneumonia from other pathogens, and analysed them using flow cytometry and bulk transcriptomic profiling. We performed single-cell RNA sequencing on 10 bronchoalveolar lavage fluid samples collected from patients with severe coronavirus disease 2019 (COVID-19) within 48 h of intubation. In the majority of patients with SARS-CoV-2 infection, the alveolar space was persistently enriched in T cells and monocytes. Bulk and single-cell transcriptomic profiling suggested that SARS-CoV-2 infects alveolar macrophages, which in turn respond by producing T cell chemoattractants. These T cells produce interferon-γ to induce inflammatory cytokine release from alveolar macrophages and further promote T cell activation. Collectively, our results suggest that SARS-CoV-2 causes a slowly unfolding, spatially limited alveolitis in which alveolar macrophages containing SARS-CoV-2 and T cells form a positive feedback loop that drives persistent alveolar inflammation.

Associations of Social Vulnerability Index With Pathologic Myocardial Findings at Autopsy.

Background: Social vulnerability is an important determinant of cardiovascular health. Prior investigations have shown strong associations of social determinants of health with cardiovascular risk factors, imaging findings, and clinical events. However, limited data exist regarding the potential role of social vulnerability and related physiologic stressors on tissue-level pathology. Methods: We analyzed clinical data and linked autopsy reports from 853 decedent individuals who underwent autopsy from 4/6/2002 to 4/1/2021 at a large urban medical center. The mean age at death was 62.9 (SD = 15.6) and 49% of decedent individuals were men. The primary exposure was census-tract level composite social vulnerability index based on the Centers for Disease Control and Prevention Social Vulnerability Index (SVI). Individuals were geocoded to census tracts and assigned SVI accordingly. Four myocardial tissue-level outcomes from autopsy were recorded as present or absent: any coronary atherosclerosis, severe/obstructive coronary atherosclerosis, myocardial fibrosis, and/or myopericardial inflammation. Multivariable-adjusted logistic regression models were constructed with SVI as the primary exposure and covariates including age, sex, race, body mass index (BMI), diabetes, and hypertension. Additional analyses were performed stratified by clinical diagnoses of heart failure (HF) and coronary artery disease (CAD). Results: In the overall cohort, SVI was not associated with outcomes on cardiac pathology in multivariable-adjusted models. However, in stratified multivariable-adjusted analyses, higher SVI (higher social vulnerability) was associated with a higher odds of myocardial fibrosis among individuals without clinical diagnoses of HF. Conclusions: Higher indices of social vulnerability are associated with a higher odds of myocardial fibrosis at autopsy among individuals without known clinical diagnoses of HF. Potential pathophysiological mechanisms and implications for prevention/treatment of myocardial dysfunction require further study.

Lung transplantation for patients with severe COVID-19.

Lung transplantation can potentially be a life-saving treatment for patients with nonresolving COVID-19-associated respiratory failure. Concerns limiting lung transplantation include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and the potential risk for allograft infection by pathogens causing ventilator-associated pneumonia in the native lung. Additionally, the native lung might recover, resulting in long-term outcomes preferable to those of transplant. Here, we report the results of lung transplantation in three patients with nonresolving COVID-19-associated respiratory failure. We performed single-molecule fluorescence in situ hybridization (smFISH) to detect both positive and negative strands of SARS-CoV-2 RNA in explanted lung tissue from the three patients and in additional control lung tissue samples. We conducted extracellular matrix imaging and single-cell RNA sequencing on explanted lung tissue from the three patients who underwent transplantation and on warm postmortem lung biopsies from two patients who had died from COVID-19-associated pneumonia. Lungs from these five patients with prolonged COVID-19 disease were free of SARS-CoV-2 as detected by smFISH, but pathology showed extensive evidence of injury and fibrosis that resembled end-stage pulmonary fibrosis. Using machine learning, we compared single-cell RNA sequencing data from the lungs of patients with late-stage COVID-19 to that from the lungs of patients with pulmonary fibrosis and identified similarities in gene expression across cell lineages. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is their only option for survival.

Lung transplantation for pulmonary fibrosis secondary to severe COVID-19.

Lung transplantation can potentially be a life-saving treatment for patients with non-resolving COVID-19 acute respiratory distress syndrome. Concerns limiting transplant include recurrence of SARS-CoV-2 infection in the allograft, technical challenges imposed by viral-mediated injury to the native lung, and potential risk for allograft infection by pathogens associated with ventilator-induced pneumonia in the native lung. Additionally, the native lung might recover, resulting in long-term outcomes preferable to transplant. Here, we report the results of the first two successful lung transplantation procedures in patients with non-resolving COVID-19 associated acute respiratory distress syndrome in the United States. We performed smFISH to detect both positive and negative strands of SARS-CoV-2 RNA in the explanted lung tissue, extracellular matrix imaging using SHIELD tissue clearance, and single cell RNA-Seq on explant and warm post-mortem lung biopsies from patients who died from severe COVID-19 pneumonia. Lungs from patients with prolonged COVID-19 were free of virus but pathology showed extensive evidence of injury and fibrosis which resembled end-stage pulmonary fibrosis. Single cell RNA-Seq of the explanted native lungs from transplant and paired warm post-mortem autopsies showed similarities between late SARS-CoV-2 acute respiratory distress syndrome and irreversible end-stage pulmonary fibrosis requiring lung transplantation. There was no recurrence of SARS-CoV-2 or pathogens associated with pre-transplant ventilator associated pneumonias following transplantation in either patient. Our findings suggest that some patients with severe COVID-19 develop fibrotic lung disease for which lung transplantation is the only option for survival. SINGLE SENTENCE SUMMARY: Some patients with severe COVID-19 develop end-stage pulmonary fibrosis for which lung transplantation may be the only treatment.