Urinary angiotensin-converting enzyme 2 and metabolomics in COVID-19-mediated kidney injury.

Background: Angiotensin-converting enzyme 2 (ACE2), the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is highly expressed in the kidneys. Beyond serving as a crucial endogenous regulator of the renin-angiotensin system, ACE2 also possess a unique function to facilitate amino acid absorption. Our observational study sought to explore the relationship between urine ACE2 (uACE2) and renal outcomes in coronavirus disease 2019 (COVID-19). Methods: In a cohort of 104 patients with COVID-19 without acute kidney injury (AKI), 43 patients with COVID-19-mediated AKI and 36 non-COVID-19 controls, we measured uACE2, urine tumour necrosis factor receptors I and II (uTNF-RI and uTNF-RII) and neutrophil gelatinase-associated lipocalin (uNGAL). We also assessed ACE2 staining in autopsy kidney samples and generated a propensity score-matched subgroup of patients to perform a targeted urine metabolomic study to describe the characteristic signature of COVID-19. Results: uACE2 is increased in patients with COVID-19 and further increased in those that developed AKI. After adjusting uACE2 levels for age, sex and previous comorbidities, increased uACE2 was independently associated with a >3-fold higher risk of developing AKI [odds ratio 3.05 (95% confidence interval 1.23‒7.58), P = .017]. Increased uACE2 corresponded to a tubular loss of ACE2 in kidney sections and strongly correlated with uTNF-RI and uTNF-RII. Urine quantitative metabolome analysis revealed an increased excretion of essential amino acids in patients with COVID-19, including leucine, isoleucine, tryptophan and phenylalanine. Additionally, a strong correlation was observed between urine amino acids and uACE2. Conclusions: Elevated uACE2 is related to AKI in patients with COVID-19. The loss of tubular ACE2 during SARS-CoV-2 infection demonstrates a potential link between aminoaciduria and proximal tubular injury.

Autopsies Revealed Pathological Features of COVID-19 in Unvaccinated vs. Vaccinated Patients.

Background: In Italy, by the end of 2021, a new pandemic wave led to increased hospitalizations and death, even in some vaccinated people. We aimed to investigate the death of COVID-19-vaccinated patients who acquired infection and developed severe disease, and to assess differences with fatal COVID-19 in unvaccinated subjects by studying the pathological events triggered by SARS-CoV-2. Methods: Detailed autoptic examination was performed on five fully vaccinated compared to five unvaccinated patients. Histopathological analysis focused on the lung and heart, the two major affected organs. Results: COVID-19 caused, or contributed to death, in all the unvaccinated cases. By contrast, in vaccinated group, pre-existing pathologies played a major role, and death was not COVID-19-related in four out of five patients. These patients did not show the histological features of SARS-CoV-2 lung damage. Diffuse inflammatory macrophages infiltration recently emerged as the main feature of COVID-19 cardiac injury. Interestingly, the most striking difference between the two groups was the absence of increased macrophage infiltration in the heart of vaccinated patients. Conclusions: Results of this study confirm the efficacy of anti-SARS-CoV-2 vaccination in protecting organs from injury and support the need to maintain an adequate immune response by booster dose administration.

SARS-CoV-2 infection of thymus induces loss of function that correlates with disease severity.

BACKGROUND: Lymphopenia, particularly when restricted to the T-cell compartment, has been described as one of the major clinical hallmarks in patients with coronavirus disease 2019 (COVID-19) and proposed as an indicator of disease severity. Although several mechanisms fostering COVID-19-related lymphopenia have been described, including cell apoptosis and tissue homing, the underlying causes of the decline in T-cell count and function are still not completely understood. OBJECTIVE: Given that viral infections can directly target thymic microenvironment and impair the process of T-cell generation, we sought to investigate the impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on thymic function. METHODS: We performed molecular quantification of T-cell receptor excision circles and κ-deleting recombination excision circles to assess, respectively, T- and B-cell neogenesis in SARS-CoV-2-infected patients. We developed a system for in vitro culture of primary human thymic epithelial cells (TECs) to mechanistically investigate the impact of SARS-CoV-2 on TEC function. RESULTS: We showed that patients with COVID-19 had reduced thymic function that was inversely associated with the severity of the disease. We found that angiotensin-converting enzyme 2, through which SARS-CoV-2 enters the host cells, was expressed by thymic epithelium, and in particular by medullary TECs. We also demonstrated that SARS-CoV-2 can target TECs and downregulate critical genes and pathways associated with epithelial cell adhesion and survival. CONCLUSIONS: Our data demonstrate that the human thymus is a target of SARS-CoV-2 and thymic function is altered following infection. These findings expand our current knowledge of the effects of SARS-CoV-2 infection on T-cell homeostasis and suggest that monitoring thymic activity may be a useful marker to predict disease severity and progression.

SARS-CoV-2 infection downregulates myocardial ACE2 and potentiates cardiac inflammation in humans and hamsters.

Myocardial pathologies resulting from SARS-CoV-2 infections are consistently rising with mounting case rates and reinfections; however, the precise global burden is largely unknown and will have an unprecedented impact. Understanding the mechanisms of COVID-19-mediated cardiac injury is essential toward the development of cardioprotective agents that are urgently needed. Assessing novel therapeutic strategies to tackle COVID-19 necessitates an animal model that recapitulates human disease. Here, we sought to compare SARS-CoV-2-infected animals with patients with COVID-19 to identify common mechanisms of cardiac injury. Two-month-old hamsters were infected with either the ancestral (D614) or Delta variant (B.1.617.2) of SARS-CoV-2 for 2 days, 7 days, and/or 14 days. We measured viral RNA and cytokine expression at the earlier time points to capture the initial stages of infection in the lung and heart. We assessed myocardial angiotensin-converting enzyme 2 (ACE2), the entry receptor for the SARS-CoV-2 virus, and cardioprotective enzyme, as well as markers for inflammatory cell infiltration in the hamster hearts at days 7 and 14. In parallel, human hearts were stained for ACE2, viral nucleocapsid, and inflammatory cells. Indeed, we identify myocardial ACE2 downregulation and myeloid cell burden as common events in both hamsters and humans infected with SARS-CoV-2, and we propose targeting downstream ACE2 downregulation as a therapeutic avenue that warrants clinical investigation.NEW & NOTEWORTHY Cardiac manifestations of COVID-19 in humans are mirrored in the SARS-CoV-2 hamster model, recapitulating myocardial damage, ACE2 downregulation, and a consistent pattern of immune cell infiltration independent of viral dose and variant. Therefore, the hamster model is a valid approach to study therapeutic strategies for COVID-19-related heart disease.

Pleural Mesothelial Cells Modulate the Inflammatory/Profibrotic Response During SARS-CoV-2 Infection.

Although lung fibrosis has a major impact in COVID-19 disease, its pathogenesis is incompletely understood. In particular, no direct evidence of pleura implication in COVID-19-related fibrotic damage has been reported so far. In this study, the expression of epithelial cytokeratins and Wilms tumor 1 (WT1), specific markers of mesothelial cells (MCs), was analyzed in COVID-19 and unrelated pleura autoptic samples. SARS-CoV-2 replication was analyzed by RT-PCR and confocal microscopy in MeT5A, a pleura MC line. SARS-CoV-2 receptors were analyzed by RT-PCR and western blot. Inflammatory cytokines from the supernatants of SARS-CoV-2-infected MeT5A cells were analysed by Luminex and ELLA assays. Immunohistochemistry of COVID-19 pleura patients highlighted disruption of pleura monolayer and fibrosis of the sub-mesothelial stroma, with the presence of MCs with fibroblastoid morphology in the sub-mesothelial stroma, but no evidence of direct infection in vivo. Interestingly, we found evidence of ACE2 expression in MCs from pleura of COVID-19 patients. In vitro analysis shown that MeT5A cells expressed ACE2, TMPRSS2, ADAM17 and NRP1, plasma membrane receptors implicated in SARS-CoV-2 cell entry and infectivity. Moreover, MeT5A cells sustained SARS-CoV-2 replication and productive infection. Infected MeT5A cells produced interferons, inflammatory cytokines and metalloproteases. Overall, our data highlight the potential role of pleura MCs as promoters of the fibrotic reaction and regulators of the immune response upon SARS-CoV-2 infection.

Dysregulation of ACE (Angiotensin-Converting Enzyme)-2 and Renin-Angiotensin Peptides in SARS-CoV-2 Mediated Mortality and End-Organ Injuries.

ACE (angiotensin-converting enzyme)-2 as the target for SARS-CoV-2 also negatively regulates the renin-angiotensin system. Pathological activation of ADAM17 (A disintegrin and metalloproteinase-17) may potentiate inflammation and diminish ACE2-mediated tissue protection through proteolytic shedding, contributing to SARS-CoV-2 pathogenesis. We aim to examine plasma soluble ACE2 and angiotensin profiles in relation to outcomes by enrolling consecutive patients admitted for COVID-19 with baseline blood collection at admission and repeated sampling at 7 days. The primary outcome was 90-day mortality, and secondary outcomes were the incidence of end-organ injuries. Overall, 242 patients were included, the median age was 63 (52-74) years, 155 (64.0%) were men, and 57 (23.6%) patients reached the primary end point. Baseline soluble ACE2 was elevated in COVID-19 but was not associated with disease severity or mortality. In contrast, an upward trajectory of soluble ACE2 at repeat sampling was independently associated with an elevated risk of mortality and incidence of acute myocardial injury and circulatory shock. Similarly, an increase in soluble tumor necrosis factor receptor levels was also associated with adverse outcomes. Plasma Ang I, Ang 1-7 (angiotensin 1-7) levels, and the Ang 1-7/Ang II (angiotensin II) ratio were elevated during SARS-CoV-2 infection related to downregulation of ACE activity at baseline. Moreover, patients having an upward trajectory of soluble ACE2 were characterized by an imbalance in the Ang 1-7/Ang II ratio. The observed dysregulation of ACE2 and angiotensin peptides with disease progression suggest a potential role of ADAM17 inhibition and enhancing the beneficial Ang 1-7/Mas axis to improve outcomes against SARS-CoV-2 infection.

May macroglossia in COVID-19 be related not only to angioedema?

SARS-CoV-2 infection can lead to a variety of clinical manifestations. The occurrence of tongue swelling has recently reported in severe cases of COVID-19, and angioedema has suggested as the causative mechanism. Several factors, such as genetic predisposing factor and angiotensin-converting enzyme inhibitors (ACEI) therapies, have proposed to induce angioedema, especially as concerns patients requiring ICU treatments. Nevertheless, the question is still debated and other causes not yet recognized should be considered. Here we present a case of macroglossia occurred in a patient deceased for COVID-19 disease, who had no family history of angioedema and did not receive ACEI as antihypertensive drug. Histological and immune-histochemical analysis revealed tongue muscle atrophy with infiltrating macrophages suggesting repair mechanisms, as seen in nerve injury recovery. These new pathological findings may open new fields of study on the pathogenesis of SARS-CoV-2.

Neuropathology and Inflammatory Cell Characterization in 10 Autoptic COVID-19 Brains.

COVID-19 presents with a wide range of clinical neurological manifestations. It has been recognized that SARS-CoV-2 infection affects both the central and peripheral nervous system, leading to smell and taste disturbances; acute ischemic and hemorrhagic cerebrovascular disease; encephalopathies and seizures; and causes most surviving patients to have long lasting neurological symptoms. Despite this, typical neuropathological features associated with the infection have still not been identified. Studies of post-mortem examinations of the cerebral cortex are obtained with difficulty due to laboratory safety concerns. In addition, they represent cases with different neurological symptoms, age or comorbidities, thus a larger number of brain autoptic data from multiple institutions would be crucial. Histopathological findings described here are aimed to increase the current knowledge on neuropathology of COVID-19 patients. We report post-mortem neuropathological findings of ten COVID-19 patients. A wide range of neuropathological lesions were seen. The cerebral cortex of all patients showed vascular changes, hyperemia of the meninges and perivascular inflammation in the cerebral parenchyma with hypoxic neuronal injury. Perivascular lymphocytic inflammation of predominantly CD8-positive T cells mixed with CD68-positive macrophages, targeting the disrupted vascular wall in the cerebral cortex, cerebellum and pons were seen. Our findings support recent reports highlighting a role of microvascular injury in COVID-19 neurological manifestations.

Dying "from" or "with" COVID-19 during the Pandemic: Medico-Legal Issues According to a Population Perspective.

There is still a lack of knowledge concerning the pathophysiology of death among COVID-19-deceased patients, and the question of whether a patient has died with or due to COVID-19 is still very much debated. In Italy, all deaths of patients who tested positive for SARS-CoV-2 are defined as COVID-19-related, without considering pre-existing diseases that may either contribute to or even cause death. Our study included nine subjects from two different nursing homes (Cases 1-4, Group A; Cases 5-9, Group B). The latter included patients who presumably died from CO poisoning due to a heating system malfunction. All subjects tested positive for COVID-19 both ante- and post-mortem and were examined using post-mortem computed tomography prior to autopsy. COVID-19 was determined to be a contributing cause in the deaths of four out of nine subjects (death due to COVID-19; i.e., pneumonia and sudden cardiac death). In the other five cases, for which CO poisoning was identified as the cause of death, the infection presumably had no role in exitus (death with COVID-19). In our attempt to classify our patients as dying with or due to COVID-19, we found the use of complete assessments (both histological analyses and computed tomography examination) fundamental.

Hepatic Failure in COVID-19: Is Iron Overload the Dangerous Trigger?

Liver injury in COVID-19 patients has progressively emerged, even in those without a history of liver disease, yet the mechanism of liver pathogenicity is still controversial. COVID-19 is frequently associated with increased serum ferritin levels, and hyperferritinemia was shown to correlate with illness severity. The liver is the major site for iron storage, and conditions of iron overload have been established to have a pathogenic role in development of liver diseases. We presented here six patients who developed severe COVID-19, with biochemical evidence of liver failure. Three cases were survived patients, who underwent liver biopsy; the other three were deceased patients, who were autopsied. None of the patients suffered underlying liver pathologies. Histopathological and ultrastructural analyses were performed. The most striking finding we demonstrated in all patients was iron accumulation into hepatocytes, associated with degenerative changes. Abundant ferritin particles were found enclosed in siderosomes, and large aggregates of hemosiderin were found, often in close contact with damaged mitochondria. Iron-caused oxidative stress may be responsible for mitochondria metabolic dysfunction. In agreement with this, association between mitochondria and lipid droplets was also found. Overall, our data suggest that hepatic iron overload could be the pathogenic trigger of liver injury associated to COVID-19.

Transglutaminase 2 Regulates Innate Immunity by Modulating the STING/TBK1/IRF3 Axis.

We have recently shown that type 2 transglutaminase (TG2) plays a key role in the host's inflammatory response during bacterial infections. In this study, we investigated whether the enzyme is involved in the regulation of the STING pathway, which is the main signaling activated in the presence of both self- and pathogen DNA in the cytoplasm, leading to type I IFN (IFN I) production. In this study, we demonstrated that TG2 negatively regulates STING signaling by impairing IRF3 phosphorylation in bone marrow-derived macrophages, isolated from wild-type and TG2 knockout mice. In the absence of TG2, we found an increase in the IFN-ß production and in the downstream JAK/STAT pathway activation. Interestingly, proteomic analysis revealed that TG2 interacts with TBK1, affecting its interactome composition. Indeed, TG2 ablation facilitates the TBK1-IRF3 interaction, thus indicating that the enzyme plays a negative regulatory effect on IRF3 recruitment in the STING/TBK1 complex. In keeping with these findings, we observed an increase in the IFNß production in bronchoalveolar lavage fluids from COVID-19-positive dead patients paralleled by a dramatic decrease of the TG2 expression in the lung pneumocytes. Taken together, these results suggest that TG2 plays a negative regulation on the IFN-ß production associated with the innate immunity response to the cytosolic presence of both self- and pathogen DNA.

Elevated P-Selectin in Severe Covid-19: Considerations for Therapeutic Options.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is mainly a respiratory tract disease and acute respiratory failure with diffuse microvascular pulmonary thrombosis are critical aspects of the morbidity and mortality of this new syndrome. PURPOSE: The aim of our study was to investigate, in severe COVID-19 hospitalized patients, the P-selectin plasma concentration as a biomarker of endothelial dysfunction and platelet activation. METHODS: 46 patients with severe or critical SARS-CoV-2 infection were included in the study. Age-matched patients then were divided in those requiring admission to the intensive care unit (ICU, ICU cases) vs those not requiring ICU hospitalization (non-ICU cases). Blood samples of severe COVID-19 patients were collected at the time of hospital admission. The quantification of soluble P-selectin was performed by ELI, assay. RESULTS: Our study showed a higher P-selectin plasma concentration in patients with Covid-19, regardless of ICU admission, compared to the normal reference values and compared to ten contextually sampled healthy donors (HD); (COVID-19): median 65.2 (IQRs: 45.1-81.1) vs. HD: 40.3 (IQRs: 24.3-48.7), p=0023. Moreover, results showed a significant reduction of P-sele din after platelets removal in HD, in contrast, both ICU and non-ICU COVID-19 patients showed similar high levels of P-selectin with and without platelets. CONCLUSION: Elevation of P-selectin suggests a central role of platelet endothelium interaction as part of the multifaced pathogenic mechanism of COVID-19 leading to the local activation of hemostatic system forming pulmonary thrombi. Further work is necessary to determine the therapeutic role of antiplatelets agents or of the anti P-selectin antibody Crizanlizumab.

Evidences for lipid involvement in SARS-CoV-2 cytopathogenesis.

The pathogenesis of SARS-CoV-2 remains to be completely understood, and detailed SARS-CoV-2 cellular cytopathic effects requires definition. We performed a comparative ultrastructural study of SARS-CoV-1 and SARS-CoV-2 infection in Vero E6 cells and in lungs from deceased COVID-19 patients. SARS-CoV-2 induces rapid death associated with profound ultrastructural changes in Vero cells. Type II pneumocytes in lung tissue showed prominent altered features with numerous vacuoles and swollen mitochondria with presence of abundant lipid droplets. The accumulation of lipids was the most striking finding we observed in SARS-CoV-2 infected cells, both in vitro and in the lungs of patients, suggesting that lipids can be involved in SARS-CoV-2 pathogenesis. Considering that in most cases, COVID-19 patients show alteration of blood cholesterol and lipoprotein homeostasis, our findings highlight a peculiar important topic that can suggest new approaches for pharmacological treatment to contrast the pathogenicity of SARS-CoV-2.

Dermatological manifestations during COVID-19 and histological picture: Description of two clinical cases.

It is not yet entirely clear what is the relevance of skin symptoms and what clinical implications are related to their appearance in COVID-19 patients. We describe two cases of COVID-19-associated pneumonia, which presented skin manifestations in advanced stage of illness, when nasopharyngeal swabs became negative for SARS-CoV-2. The first case presented erythematous, maculopapular lesions; the second developed petechial, vesicular and blood-encrusted lesions on the limbs. Histopathology documented perivascular lymphocytic infiltrates, with prevalent CD4+ T-cells in both patients. The research of SARS-CoV-2 in tissues with real time RT-PCR was negative. Basal keratinocytes displayed C4d deposits in one case, who developed laboratory signs indicative of a procoagulative condition at the same time as the skin rash. Skin manifestations during SARS-CoV-2 infection seem to be clinically relevant and further studies are necessary to assess if they are linked to systemic complications, lack of viral clearance or cascades of immune responses induced by the virus, even in patients affected by mild pneumonia.

Fatal pulmonary arterial thrombosis in a COVID-19 patient, with asymptomatic history, occurred after swab negativization.

BACKGROUND: A considerable number of SARS-CoV-2 infected individuals could be asymptomatic and don't need medical treatment. The clinical spectrum of SARS-CoV-2 infection ranges from asymptomatic cases, medium-intensity forms with mild to moderate symptoms, to severe ones with bilateral pneumonia and respiratory distress. In cases with severe presentation of SARS-CoV-2 infection, the induction of hypercoagulability is one of the pathophysiological mechanism that can contribute to death. CASE PRESENTATION: Here, we reported autoptic evidences of thrombotic pulmonary arterial fatal lesions in an asymptomatic COVID-19 patient, after swab negativization. Whole body complete post-mortem examination was performed, showing the presence of a large thrombus occluding the main pulmonary artery that was the cause of death. Histopathological analysis showed heterogeneous pattern of pathological changes in the lung tissue with numerous vascular thrombi, inflammatory cardiomyopathy and other histopathological modifications in kidneys, spleen and liver. CONCLUSIONS: This study provides evidences that also asymptomatic patients may be at risk to develop thrombotic complications. An appropriate diagnostic screening for thrombotic complications and the early treatment recommendations of antithrombotic drugs could represent an important topic even in asymptomatic individuals.

Fatal Takotsubo syndrome in critical COVID-19 related pneumonia.

COVID-19 can involve several organs and systems, often with indirect and poorly clarified mechanisms. Different presentations of myocardial injury have been reported, with variable degrees of severity, often impacting on the prognosis of COVID-19 patients. The pathogenic mechanisms underlying cardiac damage in SARS-CoV-2 infection are under active investigation. We report the clinical and autopsy findings of a fatal case of Takotsubo Syndrome occurring in an 83-year-old patient with COVID-19 pneumonia. The patient was admitted to Emergency Department with dyspnea, fever and diarrhea. A naso-pharyngeal swab test for SARS-CoV-2 was positive. In the following week his conditions worsened, requiring intubation and deep sedation. While in the ICU, the patient suddenly showed ST segment elevation. Left ventricular angiography showed decreased with hypercontractile ventricular bases and mid-apical ballooning, consistent with diagnosis of Takotsubo syndrome. Shortly after the patient was pulseless. After extensive resuscitation maneuvers, the patient was declared dead. Autopsy revealed a subepicardial hematoma, in absence of myocardial rupture. On histology, the myocardium showed diffuse edema, multiple foci of contraction band necrosis in both ventricles and occasional coagulative necrosis of single cardiac myocytes. Abundant macrophages CD68+ were detected in the myocardial interstitium. The finding of diffuse contraction band necrosis supports the pathogenic role of increased catecholamine levels; the presence of a significant interstitial inflammatory infiltrate, made up by macrophages, remains of uncertain significance.

Postmortem Findings in Italian Patients With COVID-19: A Descriptive Full Autopsy Study of Cases With and Without Comorbidities.

BACKGROUND: Descriptions of the pathological features of coronavirus disease-2019 (COVID-19) caused by the novel zoonotic pathogen severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) emanate from tissue biopsies, case reports, and small postmortem studies restricted to the lung and specific organs. Whole-body autopsy studies of COVID-19 patients have been sparse. METHODS: To further define the pathology caused by SARS-CoV-2 across all body organs, we performed autopsies on 22 patients with COVID-19 (18 with comorbidities and 4 without comorbidities) who died at the National Institute for Infectious Diseases Lazzaro Spallanzani-IRCCS Hospital, Rome, Italy. Tissues from the lung, heart, liver, kidney, spleen, and bone marrow (but not the brain) were examined. Only lung tissues were subject to transmission electron microscopy. RESULTS: COVID-19 caused multisystem pathology. Pulmonary and cardiovascular involvement were dominant pathological features. Extrapulmonary manifestations included hepatic, kidney, splenic, and bone marrow involvement, and microvascular injury and thrombosis were also detected. These findings were similar in patients with or without preexisting medical comorbidities. CONCLUSIONS: SARS-CoV-2 infection causes multisystem disease and significant pathology in most organs in patients with and without comorbidities.