Drug repurposing and other strategies for rapid antiviral development: lessons from the early stage of the pandemic

The recent and recurrent spillover of three highly pathogenic coronaviruses, SARS-CoV-1, MERS-CoV and SARS-CoV-2, into human populations has stressed the importance of pandemic preparedness. Here, we describe how, in the absence of antiviral therapeutic options against coronaviruses, early clinical investigations have focused on the prompt repurposing of approved antiviral therapies. We discuss how, despite international collaborative efforts, their outcomes so far have been disappointing as none of the early drugs tested demonstrated effective clinical efficacy. We also outline innovative strategies and tools developed to fast-track development of novel classes of antivirals. These capitalise on a deeper understanding of viral molecular pathogenesis and how coronaviruses hijack the host cellular machinery to maximise their replication and counteract host defences. Collectively, these approaches are crucial to identify and validate novel targets for therapeutic interventions and expand the repertoire of broad-spectrum antiviral agents, so that these can be promptly deployed for current and future pandemics.Copyright © ERS 2021.

4th Global Conference on Myositis, GCOM

The proceedings contain 255 papers. The topics discussed include: molecular pathology of mitochondrial disorders;defining causal genes at MHC in SLE - implications for myositis and other diseases that share MHC risk;role of mitochondria in skeletal muscle dysfunction in myositis;selective T cell depletion for inclusion body myositis: why and how;inclusion body myositis in 2022: from physiopathogenesis to clinical trials;autoantibodies and complement in experimental IMNM: from pathogenesis to therapy?;reliability of immunoassays for myositis autoantibodies;when JM patients lose their 'J': transition challenges in myositis car;fatigue and well-being of children with chronic inflammatory disease;physical fitness in long-term JDM;Eular Covid and COVAX registries' update: focus on myositis;and outcomes, biomarkers, and novel treatments for the skin in dermatomyositis.

C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps.

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.

T-Cell Immunity in COVID-19-Recovered Individuals and Individuals Vaccinated with the Combined Vector Vaccine Gam-COVID-Vac.

The COVID-19 pandemic has required extensive research on the new coronavirus SARS-CoV-2 and the creation of new highly effective vaccines. The presence of T-cells in the body that respond to virus antigens suggests adequate antiviral immunity. We investigated T-cell immunity in individuals who recovered from mild and moderate COVID-19 and in individuals vaccinated with the Gam-COVID-Vac combined vector vaccine. The ELISPOT method was used to determine the number of T-cells responding with IFN-γ synthesis to stimulation by peptides containing epitopes of the S-protein or N-, M-, ORF3, and ORF7 proteins, using peripheral blood mononuclear cells (PBMCs). At the same time, the multiplex method was used to determine the accumulation of IFN-γ and other cytokines in the culture medium. According to the data obtained, the proportion of positive conclusions about the T-cell immune response to SARS-CoV-2 antigens in control, recovered, and vaccinated individuals was 12%, 70%, and 52%, respectively. At the same time, more than half of the vaccinated individuals with a T-cell response were sensitized to the antigens of N-, M-, ORF3, and ORF7 proteins not produced by Gam-COVID-Vac, indicating a high likelihood of asymptomatic SARS-CoV-2 infection. Increased IFN-γ release by single sensitized T-cells in response to specific stimulation in recovered and vaccinated individuals did not result in the accumulation of this and other cytokines in the culture medium. These findings suggest a balance between cytokine production and utilization by immunocompetent cells as a prerequisite for providing a controlled cytokine signal and avoiding a "cytokine storm".

[Massive pancarditis-autopsy report]. / Massive Pankarditis ­ ein Autopsiebericht.

We report on a 69-year-old man suffering from chronic progressive oligoarthritis (localized in metacarpal and knee joints), which clinically was interpreted as steroid-sensitive seronegative chronic arthritis. The patient died from sudden death at the emergency department after a 4-week history of increasing cough and dyspnea (meanwhile obtaining negative testing results for SARS-CoV-2). During the autopsy, we found massive pancarditis affecting all cardiac compartments, in particular exhibiting constrictive pericarditis, myocarditis, and multivalvular endocarditis. Microscopically, interstitial myocarditis could be observed. Performing extensive molecular analyses, we detected Tropheryma whipplei in the tissue specimens of the heart, but not in various duodenal tissue probes or in the synovial membrane. Taken together, in the present case the cause of death was acute cardiac failure due to multivalvular pancarditis due to T. whipplei. Besides from classical symptoms and morphological signs, Whipple's disease may present with various features. Regarding the differential diagnosis of a chronic multisystem disorder with aspects of hitherto unknown arthralgia, Whipple's disease should be considered.

A specific molecular signature in SARS-CoV-2-infected kidney biopsies.

Acute kidney injury is one of the most important complications in patients with COVID-19 and is considered a negative prognostic factor with respect to patient survival. The occurrence of direct infection of the kidney by SARS-CoV-2, and its contribution to the renal deterioration process, remain controversial issues. By studying 32 renal biopsies from patients with COVID-19, we verified that the major pathological feature of COVID-19 is acute tubular injury (ATI). Using single-molecule fluorescence in situ hybridization, we showed that SARS-CoV-2 infected living renal cells and that infection, which paralleled renal angiotensin-converting enzyme 2 expression levels, was associated with increased death. Mechanistically, a transcriptomic analysis uncovered specific molecular signatures in SARS-CoV-2-infected kidneys as compared with healthy kidneys and non-COVID-19 ATI kidneys. On the other hand, we demonstrated that SARS-CoV-2 and hantavirus, 2 RNA viruses, activated different genetic networks despite triggering the same pathological lesions. Finally, we identified X-linked inhibitor of apoptosis-associated factor 1 as a critical target of SARS-CoV-2 infection. In conclusion, this study demonstrated that SARS-CoV-2 can directly infect living renal cells and identified specific druggable molecular targets that can potentially aid in the design of novel therapeutic strategies to preserve renal function in patients with COVID-19.

Spatial transcriptomic characterization of COVID-19 pneumonitis identifies immune circuits related to tissue injury.

Severe lung damage in COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways and genes present across the spectrum of histopathological damage in COVID-19 lung tissue. We applied correlation network-based approaches to deconvolve gene expression data from 46 areas of interest covering >62,000 cells within well preserved lung samples from three patients. Despite substantial inter-patient heterogeneity, we discovered evidence for a common immune cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines including CXCL9, CXCL10 and CXCL11 which are known to promote the recruitment of CXCR3+ immune cells. The tumour necrosis factor (TNF) superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were found to be consistently upregulated in the areas with severe tissue damage. We used published spatial and single cell SARS-CoV-2 datasets to confirm our findings in the lung tissue from additional cohorts of COVID-19 patients. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

Toward a More Just System of Care in Molecular Pathology.

Policy Points American health care policy must be critically assessed to establish the role it plays in sustaining and alleviating the health disparities that currently exist in molecular genetic testing. It is critical to understand the economic and sociocultural influences that drive patients to undergo or forgo molecular testing, especially in marginalized patient populations. A multipronged solution with actions necessary from multiple stakeholders is required to reduce the cost of health care, rebalance regional disparities, encourage physician engagement, reduce data bias, and earn patients' trust. CONTEXT: The health status of a population is greatly influenced by both biological processes and external factors. For years, minority and low socioeconomic patient populations have faced worse outcomes and poorer health in the United States. Experts have worked extensively to understand the issues and find solutions to alleviate this disproportionate burden of disease. As a result, there have been some improvements and successes, but wide gaps still exist. Diagnostic molecular genetic testing and so-called personalized medicine are just now being integrated into the current American health care system. The way in which these tests are integrated can either exacerbate or reduce health disparities. METHODS: We provide case scenarios-loosely based on real-life patients-so that nonexperts can see the impacts of complex policy decisions and unintentional biases in technology without needing to understand all the intricacies. We use data to explain these findings from an extensive literature search examining both peer-reviewed and gray literature. FINDINGS: Access to diagnostic molecular genetic testing is not equitable or sufficient, owing to at least five major factors: (1) cost to the patient, (2) location, (3) lack of provider buy-in, (4) data-set bias, and (5) lack of public trust. CONCLUSIONS: Molecular genetic pathology can be made more equitable with the concerted efforts of multiple stakeholders. Confronting the five major factors identified here may help us usher in a new era of precision medicine without its discriminatory counterpart.

One year in review 2022: systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic multisystem auto-immune disease with extremely varied clinical manifestations and a complex pathogenesis. New insights in SLE about pathogenetic pathways, biomarkers, and data on clinical manifestations are progressively emerging, and new drugs and new therapeutic strategies have been proposed to improve the control of disease activity. Thus, this review is aimed to summarise the most relevant data about SLE emerged during 2021, following the previous annual review of this series. © Copyright Clinical and Experimental Rheumatology 2022.

A retrospective cohort study to assess the levels of IL-6 in COVID-19 infected patients and to evaluate its association with the severity of the disease in Jodhpur, Rajasthan

Background: The SARS-CoV-2 virus replicates rapidly, triggering a storm characterized by increased levels of cytokines such as IL-6. Such an inflammatory response causes inflammation of the respiratory system and other bodily systems, with subsequent occurrence of ARDS or respiratory failure. The estimation of IL-6 levels could be an important tool to assess disease severity in COVID-19 patients. Methodology: A retrospective cohort study was conducted on 45 patients in Department of Medicine, MDM Hospital, Dr. S.N Medical College, Jodhpur, Rajasthan. A predesigned and pre-structure Performa was used. Conclusion: IL-6 is an adequate predictor of severe disease in patients infected with the COVID-19 virus. The finding of current study guide clinicians and healthcare providers in identifying potentially severe or critical patients with COVID-19.

139th Congress of the German Society of Surgery, DGCH

The proceedings contain 32 papers. The topics discussed include: molecular pathogenesis and prognostic stratification: acute myeloid leukemia;molecular pathogenesis and prognostic stratification of myelodysplastic syndromes;extracellular vesicles and their role in tumor microenvironment;new genomic, transcriptomic and cellular risk factors in multiple myeloma;drivers of progression in MPNS;SARS-COV2 coagulopathy: an intricated puzzle;role of the microenvironment in the pathogenesis of myelodysplastic syndromes;the tumor microenvironment in Hodgkin lymphoma: clinical implications;CRISPR/CAS9-based genome editing to model the anti-leukemia immune response and identify its molecular players;and deregulation of the transforming growth factor-B pathway in low-risk MDS patients SF3B1 mutated.

Lethal synergy between SARS-CoV-2 and Streptococcus pneumoniae in hACE2 mice and protective efficacy of vaccination.

Secondary infections are frequent complications of viral respiratory infections, but the potential consequence of SARS-CoV-2 coinfection with common pulmonary pathogens is poorly understood. We report that coinfection of human ACE2-transgenic mice with sublethal doses of SARS-CoV-2 and Streptococcus pneumoniae results in synergistic lung inflammation and lethality. Mortality was observed regardless of whether SARS-CoV-2 challenge occurred before or after establishment of sublethal pneumococcal infection. Increased bacterial levels following coinfection were associated with alveolar macrophage depletion, and treatment with murine GM-CSF reduced numbers of lung bacteria and pathology and partially protected from death. However, therapeutic targeting of IFNs, an approach that is effective against influenza coinfections, failed to increase survival. Combined vaccination against both SARS-CoV-2 and pneumococci resulted in 100% protection against subsequent coinfection. The results indicate that when seasonal respiratory infections return to prepandemic levels, they could lead to an increased incidence of lethal COVID-19 superinfections, especially among the unvaccinated population.

Morphological and tissue-based molecular characterization of oral lesions in patients with COVID-19: A living systematic review.

OBJECTIVE: This living systematic review aims to integrate the morphological and tissue-based molecular characterization of oral lesions occurring in individuals infected by COVID-19 (OLICs). MATERIALS AND DESIGN: This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. PubMed, Web of Science, Scopus, Ovid, Embase, and LILACS were searched to identify reports on OLICs with morphological and/or tissue-based molecular data. RESULTS: Four studies reporting five cases were included. Three patients were male, and the mean age of the individuals was 47.6 years. The most reported anatomical location was the palate (n = 4), whereas ulcers were the most frequent clinical presentation (n = 3). Histopathologically, all cases revealed cell vacuolization and exocytosis in the epithelial layer. In the mesenchymal layer, inflammatory cell infiltrate and thrombi/microvascular thrombosis were observed in three cases. Immunohistochemical reactions were performed in two cases. Both cases were negative for HHV-1, HHV-2, and CMV. One case revealed positivity for SARS-CoV-2 spike protein. No other molecular tests were found for the characterization of OLIC. CONCLUSIONS: The pathological characteristics of OLICs are still unspecific. However, with the ongoing COVID-19 pandemic and well-documented new cases, whether OLICs are due to coinfections or has a primary origin can be determined.

Molecular pathogenesis of cardiac microthrombi in fatal covid-19: Insights from clinicohistopathologic and single nuclei rna sequencing analyses

Cardiac microthrombi are postulated to underlie cardiac injury in critical COVID-19. To determine pathogenic mechanism(s) of cardiac injury in fatal COVID-19, we conducted a single-center prospective cohort study of 69 consecutive COVID-19 decedents. Microthrombi was the most commonly detected acute cardiac histopathologic feature (n=48, 70%). We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with inhospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak ESR and CRP during hospitalization were independently associated with higher odds of microthrombi (ESR, Pnonlinearity 0.015, Passociation=0.008;CRP per 20mg/L increase, OR 1.17, 95%CI 1.00-1.36). Using single nuclei RNA-sequence analysis, we discovered an enrichment of prothrombotic, anti-fibrinolytic, and extracellular matrix signaling amongst cardiac fibroblasts in microthrombi-positive COVID-19 hearts, compared with microthrombi-negative COVID-19 hearts and non-COVID-19 donor hearts. Our cumulative findings identify these specific transcriptomic changes in cardiac fibroblasts as salient features of COVID-19-associated cardiac microthrombi.

Clinico-histopathologic and single-nuclei RNA-sequencing insights into cardiac injury and microthrombi in critical COVID-19.

Acute cardiac injury is prevalent in critical COVID-19 and associated with increased mortality. Its etiology remains debated, as initially presumed causes - myocarditis and cardiac necrosis - have proved uncommon. To elucidate the pathophysiology of COVID-19-associated cardiac injury, we conducted a prospective study of the first 69 consecutive COVID-19 decedents at CUIMC in New York City. Of 6 acute cardiac histopathologic features, presence of microthrombi was the most commonly detected among our cohort. We tested associations of cardiac microthrombi with biomarkers of inflammation, cardiac injury, and fibrinolysis and with in-hospital antiplatelet therapy, therapeutic anticoagulation, and corticosteroid treatment, while adjusting for multiple clinical factors, including COVID-19 therapies. Higher peak erythrocyte sedimentation rate and C-reactive protein were independently associated with increased odds of microthrombi, supporting an immunothrombotic etiology. Using single-nuclei RNA-sequencing analysis on 3 patients with and 4 patients without cardiac microthrombi, we discovered an enrichment of prothrombotic/antifibrinolytic, extracellular matrix remodeling, and immune-potentiating signaling among cardiac fibroblasts in microthrombi-positive, relative to microthrombi-negative, COVID-19 hearts. Non-COVID-19, nonfailing hearts were used as reference controls. Our study identifies a specific transcriptomic signature in cardiac fibroblasts as a salient feature of microthrombi-positive COVID-19 hearts. Our findings warrant further mechanistic study as cardiac fibroblasts may represent a potential therapeutic target for COVID-19-associated cardiac microthrombi.

Cross-validation of SARS-CoV-2 responses in kidney organoids and clinical populations.

Kidneys are critical target organs of COVID-19, but susceptibility and responses to infection remain poorly understood. Here, we combine SARS-CoV-2 variants with genome-edited kidney organoids and clinical data to investigate tropism, mechanism, and therapeutics. SARS-CoV-2 specifically infects organoid proximal tubules among diverse cell types. Infections produce replicating virus, apoptosis, and disrupted cell morphology, features of which are revealed in the context of polycystic kidney disease. Cross-validation of gene expression patterns in organoids reflects proteomic signatures of COVID-19 in the urine of critically ill patients indicating interferon pathway upregulation. SARS-CoV-2 viral variants alpha, beta, gamma, kappa, and delta exhibit comparable levels of infection in organoids. Infection is ameliorated in ACE2-/- organoids and blocked via treatment with de novo-designed spike binder peptides. Collectively, these studies clarify the impact of kidney infection in COVID-19 as reflected in organoids and clinical populations, enabling assessment of viral fitness and emerging therapies.

Chitinase 3-like-1 is a therapeutic target that mediates the effects of aging in COVID-19.

COVID-19 is caused by SARS-CoV-2 (SC2) and is more prevalent and severe in elderly and patients with comorbid diseases (CM). Because chitinase 3-like-1 (CHI3L1) is induced during aging and CM, the relationships between CHI3L1 and SC2 were investigated. Here, we demonstrate that CHI3L1 is a potent stimulator of the SC2 receptor angiotensin converting enzyme 2 (ACE2) and viral spike protein priming proteases (SPP), that ACE2 and SPP are induced during aging, and that anti-CHI3L1, kasugamycin, and inhibitors of phosphorylation abrogate these ACE2- and SPP-inductive events. Human studies also demonstrate that the levels of circulating CHI3L1 are increased in the elderly and patients with CM, where they correlate with COVID-19 severity. These studies demonstrate that CHI3L1 is a potent stimulator of ACE2 and SPP, that this induction is a major mechanism contributing to the effects of aging during SC2 infection, and that CHI3L1 co-opts the CHI3L1 axis to augment SC2 infection. CHI3L1 plays a critical role in the pathogenesis of and is an attractive therapeutic target in COVID-19.

The Role of the Pathologist in the Next-Generation Era of Tumor Molecular Characterization.

Current pathology practice is being shaped by the increasing complexity of modern medicine, in particular of precision oncology, and major technological advances. In the "next-generation technologies era", the pathologist has become the person responsible for the integration and interpretation of morphologic and molecular information and for the delivery of critical answers to diagnostic, prognostic and predictive queries, acquiring a prominent position in the molecular tumor boards.