Differential Gene Expression of SARS-CoV-2 Positive Bronchoalveolar Lavages: A Case Series.

BACKGROUND: Transcriptomic data on bronchoalveolar lavage (BAL) from COVID-19 patients are currently scarce. OBJECTIVES: This case series seeks to characterize the intra-alveolar immunopathology of COVID-19. METHOD: BALs were performed on 14 patients (5 COVID-19, of which 3 mild and 2 largely asymptomatic, 9 controls). Controls included asthma (n = 1), unremarkable BALs (n = 3), infections with respiratory syncytial virus (n = 1), influenza B (n = 1), and infections with other coronaviruses (n = 3). SARS-CoV-2 RNA load was measured by quantitative nucleic acid testing, while the detection of other pathogens was performed by immunofluorescence or multiplex NAT. RESULTS: Gene expression profiling showed 71 significantly downregulated and 5 upregulated transcripts in SARS-CoV-2-positive lavages versus controls. Downregulated transcripts included genes involved in macrophage development, polarization, and crosstalk (LGALS3, MARCO, ERG2, BTK, RAC1, CD83), and genes involved in chemokine signaling and immunometabolism (NUPR1, CEBPB, CEBPA, PECAM1, CCL18, PPARG, ALOX5, ALOX5AP). Upregulated transcripts featured genes involved in NK-T cell signaling (GZMA, GZMH, GNLY, PRF1, CD3G). Patients with mild COVID-19 showed a significant upregulation of genes involved in blood mononuclear cell/leukocyte function (G0S2, ANXA6, FCGR2B, ADORA3), coagulation (von Willebrand factor [VWF]), interferon response (IFRD1, IL12RB2), and a zinc metalloprotease elevated in asthma (CPA3) compared to asymptomatic cases. In-silico comparison of the 5 COVID-19 BAL cases to a published cohort of lethal COVID-19 showed a significant upregulation of "antigen processing and presentation" and "lysosome" pathways in lethal cases. CONCLUSIONS: These data underscore the heterogeneity of immune response in COVID-19. Further studies with a larger dataset are required to gain a better understanding of the hallmarks of SARS-CoV-2 immunological response.

Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Morphologic and molecular analysis of liver injury after SARS-CoV-2 vaccination reveals distinct characteristics.

BACKGROUND & AIMS: Liver injury after COVID-19 vaccination is very rare and shows clinical and histomorphological similarities with autoimmune hepatitis (AIH). Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI) and its relationship to AIH. Therefore, we compared VILI with AIH. METHODS: Formalin-fixed and paraffin-embedded liver biopsy samples from patients with VILI (n = 6) and from patients with an initial diagnosis of AIH (n = 9) were included. Both cohorts were compared by histomorphological evaluation, whole-transcriptome and spatial transcriptome sequencing, multiplex immunofluorescence, and immune repertoire sequencing. RESULTS: Histomorphology was similar in both cohorts but showed more pronounced centrilobular necrosis in VILI. Gene expression profiling showed that mitochondrial metabolism and oxidative stress-related pathways were more and interferon response pathways were less enriched in VILI. Multiplex analysis revealed that inflammation in VILI was dominated by CD8+ effector T cells, similar to drug-induced autoimmune-like hepatitis. In contrast, AIH showed a dominance of CD4+ effector T cells and CD79a+ B and plasma cells. T-cell receptor (TCR) and B-cell receptor sequencing showed that T and B cell clones were more dominant in VILI than in AIH. In addition, many T cell clones detected in the liver were also found in the blood. Interestingly, analysis of TCR beta chain and Ig heavy chain variable-joining gene usage further showed that TRBV6-1, TRBV5-1, TRBV7-6, and IgHV1-24 genes are used differently in VILI than in AIH. CONCLUSIONS: Our analyses support that SARS-CoV-2 VILI is related to AIH but also shows distinct differences from AIH in histomorphology, pathway activation, cellular immune infiltrates, and TCR usage. Therefore, VILI may be a separate entity, which is distinct from AIH and more closely related to drug-induced autoimmune-like hepatitis. IMPACT AND IMPLICATIONS: Little is known about the pathophysiology of COVID-19 vaccine-induced liver injury (VILI). Our analysis shows that COVID-19 VILI shares some similarities with autoimmune hepatitis, but also has distinct differences such as increased activation of metabolic pathways, a more prominent CD8+ T cell infiltrate, and an oligoclonal T and B cell response. Our findings suggest that VILI is a distinct disease entity. Therefore, there is a good chance that many patients with COVID-19 VILI will recover completely and will not develop long-term autoimmune hepatitis.

MicroRNA-27a-3p targets FoxO signalling to induce tumour-like phenotypes in bile duct cells.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a heterogeneous and lethal malignancy, the molecular origins of which remain poorly understood. MicroRNAs (miRs) target diverse signalling pathways, functioning as potent epigenetic regulators of transcriptional output. We aimed to characterise miRNome dysregulation in CCA, including its impact on transcriptome homeostasis and cell behaviour. METHODS: Small RNA sequencing was performed on 119 resected CCAs, 63 surrounding liver tissues, and 22 normal livers. High-throughput miR mimic screens were performed in three primary human cholangiocyte cultures. Integration of patient transcriptomes and miRseq together with miR screening data identified an oncogenic miR for characterization. MiR-mRNA interactions were investigated by a luciferase assay. MiR-CRISPR knockout cells were generated and phenotypically characterized in vitro (proliferation, migration, colony, mitochondrial function, glycolysis) and in vivo using subcutaneous xenografts. RESULTS: In total, 13% (140/1,049) of detected miRs were differentially expressed between CCA and surrounding liver tissues, including 135 that were upregulated in tumours. CCA tissues were characterised by higher miRNome heterogeneity and miR biogenesis pathway expression. Unsupervised hierarchical clustering of tumour miRNomes identified three subgroups, including distal CCA-enriched and IDH1 mutant-enriched subgroups. High-throughput screening of miR mimics uncovered 71 miRs that consistently increased proliferation of three primary cholangiocyte models and were upregulated in CCA tissues regardless of anatomical location, among which only miR-27a-3p had consistently increased expression and activity in several cohorts. FoxO signalling was predominantly downregulated by miR-27a-3p in CCA, partially through targeting of FOXO1. MiR-27a knockout increased FOXO1 levels in vitro and in vivo, impeding tumour behaviour and growth. CONCLUSIONS: The miRNomes of CCA tissues are highly remodelled, impacting transcriptome homeostasis in part through regulation of transcription factors like FOXO1. MiR-27a-3p arises as an oncogenic vulnerability in CCA. IMPACT AND IMPLICATIONS: Cholangiocarcinogenesis entails extensive cellular reprogramming driven by genetic and non-genetic alterations, but the functional roles of these non-genetic events remain poorly understood. By unveiling global miRNA upregulation in patient tumours and their functional ability to increase proliferation of cholangiocytes, these small non-coding RNAs are implicated as critical non-genetic alterations promoting biliary tumour initiation. These findings identify possible mechanisms for transcriptome rewiring during transformation, with potential implications for patient stratification.

Robust single-cell matching and multimodal analysis using shared and distinct features.

The ability to align individual cellular information from multiple experimental sources is fundamental for a systems-level understanding of biological processes. However, currently available tools are mainly designed for single-cell transcriptomics matching and integration, and generally rely on a large number of shared features across datasets for cell matching. This approach underperforms when applied to single-cell proteomic datasets due to the limited number of parameters simultaneously accessed and lack of shared markers across these experiments. Here, we introduce a cell-matching algorithm, matching with partial overlap (MARIO) that accounts for both shared and distinct features, while consisting of vital filtering steps to avoid suboptimal matching. MARIO accurately matches and integrates data from different single-cell proteomic and multimodal methods, including spatial techniques and has cross-species capabilities. MARIO robustly matched tissue macrophages identified from COVID-19 lung autopsies via codetection by indexing imaging to macrophages recovered from COVID-19 bronchoalveolar lavage fluid by cellular indexing of transcriptomes and epitopes by sequencing, revealing unique immune responses within the lung microenvironment of patients with COVID.

Genetic and epigenetic analysis of hepatocellular adenomas with atypical morphological features.

BACKGROUND: Hepatocellular adenoma (HCA) is a rare liver tumour, which can have atypical morphological features such as cytological atypia, pseudoglandular architecture, and altered reticulin framework. Little is known about the genetic and epigenetic alterations of such HCAs and whether they show the alterations classically found in hepatocellular carcinoma (HCC) or in HCA without atypical morphology. METHODS: We analysed five HCAs with atypical morphological features and one HCA with transition to HCC. Every tumour was subtyped by immunohistochemistry, sequenced by a targeted NGS panel, and analysed on a DNA methylation microarray. RESULTS: Subtyping of the five HCAs with atypical features revealed two ß-catenin mutated HCA (b-HCA), two ß-catenin mutated inflammatory HCA (b-IHCA), and one sonic hedgehog activated HCA (shHCA). None of them showed mutations typically found in HCC, such as, e.g. TERT or TP53 mutations. The epigenomic pattern of HCAs with atypical morphological features clustered with reference data for HCAs without atypical morphological features but not with HCC. Similarly, phyloepigenetic trees using the DNA methylation data reproducibly showed that HCAs with morphological atypia are much more similar to nonmalignant samples than to malignant samples. Finally, atypical HCAs showed no relevant copy number variations (CNV). CONCLUSION: In our series, mutational, DNA methylation, as well as CNV analyses, supported a relationship of atypical HCAs with nonatypical HCAs rather than with HCC. Therefore, in cases with difficult differential diagnosis between HCC and HCA, it might be advisable to perform targeted sequencing and/or combined methylation/copy number profiling.

Prevalence of Molecular Alterations in a Swiss Cohort of 512 Colorectal Carcinoma Patients by Targeted Next-Generation Sequencing Analysis in Routine Diagnostics.

INTRODUCTION: Colorectal carcinoma (CRC) is among the most common carcinomas in women and men. In the advanced stage, patients are treated based on the RAS status. Recent studies indicate that in the future, in addition to KRAS and NRAS, alterations in other genes, such as PIK3CA or TP53, will be considered for therapy. Therefore, it is important to know the mutational landscape of routinely diagnosed CRC. METHOD: We report the molecular profile of 512 Swiss CRC patients analyzed by targeted next-generation sequencing as part of routine diagnostics at our institute. RESULTS: Pathogenic and likely pathogenic variants were found in 462 (90%) CRC patients. Variants were detected in TP53 (54.3%), KRAS (48.2%), PIK3CA (15.6%), BRAF (13.5%), SMAD4 (10.5%), FBXW7 (7.8%), NRAS (3.5%), PTEN (2.7%), ERBB2 (1.6%), AKT1 (1.5%), and CTNNB1 (0.9%). The remaining pathogenic alterations were found in the genes ATM(n= 1), MAP2K1(n= 1), and IDH2(n= 1). DISCUSSION/CONCLUSIONS: Our analysis revealed the prevalence of potential predictive markers in a large cohort of CRC patients obtained during routine diagnostic analysis. Furthermore, our study is the first of this size to uncover the molecular landscape of CRC in Switzerland.

Pulmonary Surfactant Proteins Are Inhibited by Immunoglobulin A Autoantibodies in Severe COVID-19.

Rationale: Coronavirus disease 2019 (COVID-19) can lead to acute respiratory distress syndrome with fatal outcomes. Evidence suggests that dysregulated immune responses, including autoimmunity, are key pathogenic factors. Objectives: To assess whether IgA autoantibodies target lung-specific proteins and contribute to disease severity. Methods: We collected 147 blood, 9 lung tissue, and 36 BAL fluid samples from three tertiary hospitals in Switzerland and one in Germany. Severe COVID-19 was defined by the need to administer oxygen. We investigated the presence of IgA autoantibodies and their effects on pulmonary surfactant in COVID-19 using the following methods: immunofluorescence on tissue samples, immunoprecipitations followed by mass spectrometry on BAL fluid samples, enzyme-linked immunosorbent assays on blood samples, and surface tension measurements with medical surfactant. Measurements and Main Results: IgA autoantibodies targeting pulmonary surfactant proteins B and C were elevated in patients with severe COVID-19 but not in patients with influenza or bacterial pneumonia. Notably, pulmonary surfactant failed to reduce surface tension after incubation with either plasma or purified IgA from patients with severe COVID-19. Conclusions: Our data suggest that patients with severe COVID-19 harbor IgA autoantibodies against pulmonary surfactant proteins B and C and that these autoantibodies block the function of lung surfactant, potentially contributing to alveolar collapse and poor oxygenation.

Severe Neuro-COVID is associated with peripheral immune signatures, autoimmunity and neurodegeneration: a prospective cross-sectional study.

Growing evidence links COVID-19 with acute and long-term neurological dysfunction. However, the pathophysiological mechanisms resulting in central nervous system involvement remain unclear, posing both diagnostic and therapeutic challenges. Here we show outcomes of a cross-sectional clinical study (NCT04472013) including clinical and imaging data and corresponding multidimensional characterization of immune mediators in the cerebrospinal fluid (CSF) and plasma of patients belonging to different Neuro-COVID severity classes. The most prominent signs of severe Neuro-COVID are blood-brain barrier (BBB) impairment, elevated microglia activation markers and a polyclonal B cell response targeting self-antigens and non-self-antigens. COVID-19 patients show decreased regional brain volumes associating with specific CSF parameters, however, COVID-19 patients characterized by plasma cytokine storm are presenting with a non-inflammatory CSF profile. Post-acute COVID-19 syndrome strongly associates with a distinctive set of CSF and plasma mediators. Collectively, we identify several potentially actionable targets to prevent or intervene with the neurological consequences of SARS-CoV-2 infection.

SARS-CoV-2 infection drives an inflammatory response in human adipose tissue through infection of adipocytes and macrophages.

Obesity, characterized by chronic low-grade inflammation of the adipose tissue, is associated with adverse coronavirus disease 2019 (COVID-19) outcomes, yet the underlying mechanism is unknown. To explore whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection of adipose tissue contributes to pathogenesis, we evaluated COVID-19 autopsy cases and deeply profiled the response of adipose tissue to SARS-CoV-2 infection in vitro. In COVID-19 autopsy cases, we identified SARS-CoV-2 RNA in adipocytes with an associated inflammatory infiltrate. We identified two distinct cellular targets of infection: adipocytes and a subset of inflammatory adipose tissue-resident macrophages. Mature adipocytes were permissive to SARS-CoV-2 infection; although macrophages were abortively infected, SARS-CoV-2 initiated inflammatory responses within both the infected macrophages and bystander preadipocytes. These data suggest that SARS-CoV-2 infection of adipose tissue could contribute to COVID-19 severity through replication of virus within adipocytes and through induction of local and systemic inflammation driven by infection of adipose tissue-resident macrophages.

Patient-derived tumor organoids for personalized medicine in a patient with rare hepatocellular carcinoma with neuroendocrine differentiation: a case report.

Background: Hepatocellular carcinoma with neuroendocrine differentiation (HCC-NED) is a very rare subtype of primary liver cancer. Treatment allocation in these patients therefore remains a challenge. Methods: We report the case of a 74-year-old man with a HCC-NED. The tumor was surgically removed in curative intent. Histopathological work-up revealed poorly differentiated hepatocellular carcinoma (Edmondson-Steiner grade IV) with diffuse expression of neuroendocrine markers synaptophysin and chromogranin. Three months after resection, multifocal recurrence of the HCC-NED was observed. In the meantime, tumor organoids have been generated from the resected HCC-NED and extensively characterized. Sensitivity to a number of drugs approved for the treatment of HCC or neuroendocrine carcinomas was tested in vitro. Results: Based on the results of the in vitro drug screening, etoposide and carboplatin are used as first line palliative combination treatment. With genomic analysis revealing a NTRK1-mutation of unknown significance (kinase domain) and tumor organoids found to be sensitive to entrectinib, a pan-TRK inhibitor, the patient was treated with entrectinib as second line therapy. After only two weeks, treatment is discontinued due to deterioration of the patient's general condition. Conclusion: The rapid establishment of patient-derived tumor organoids allows in vitro drug testing and thereby personalized treatment choices, however clinical translation remains a challenge. To the best of our knowledge, this report provides a first proof-of-principle for using organoids for personalized medicine in this rare subtype of primary liver cancer.

Genomic analysis of focal nodular hyperplasia with associated hepatocellular carcinoma unveils its malignant potential: a case report.

Background: Focal nodular hyperplasia (FNH) is typically considered a benign tumor of the liver without malignant potential. The co-occurrence of FNH and hepatocellular carcinoma (HCC) has been reported in rare cases. In this study we sought to investigate the clonal relationship between these lesions in a patient with FNH-HCC co-occurrence. Methods: A 74-year-old female patient underwent liver tumor resection. The resected nodule was subjected to histologic analyses using hematoxylin and eosin stain and immunohistochemistry. DNA extracted from microdissected FNH and HCC regions was subjected to whole exome sequencing. Clonality analysis were performed using PyClone. Results: Histologic analysis reveals that the nodule consists of an FNH and two adjoining HCC components with distinct histopathological features. Immunophenotypic characterization and genomic analyses suggest that the FNH is clonally related to the HCC components, and is composed of multiple clones at diagnosis, that are likely to have progressed to HCC through clonal selection and/or the acquisition of additional genetic events. Conclusion: To the best of our knowledge, our work is the first study showing a clonal relationship between FNH and HCC. We show that FNH may possess the capability to undergo malignant transformation and to progress to HCC in very rare cases.

Integrative proteogenomic characterization of hepatocellular carcinoma across etiologies and stages.

Proteogenomic analyses of hepatocellular carcinomas (HCC) have focused on early-stage, HBV-associated HCCs. Here we present an integrated proteogenomic analysis of HCCs across clinical stages and etiologies. Pathways related to cell cycle, transcriptional and translational control, signaling transduction, and metabolism are dysregulated and differentially regulated on the genomic, transcriptomic, proteomic and phosphoproteomic levels. We describe candidate copy number-driven driver genes involved in epithelial-to-mesenchymal transition, the Wnt-ß-catenin, AKT/mTOR and Notch pathways, cell cycle and DNA damage regulation. The targetable aurora kinase A and CDKs are upregulated. CTNNB1 and TP53 mutations are associated with altered protein phosphorylation related to actin filament organization and lipid metabolism, respectively. Integrative proteogenomic clusters show that HCC constitutes heterogeneous subgroups with distinct regulation of biological processes, metabolic reprogramming and kinase activation. Our study provides a comprehensive overview of the proteomic and phophoproteomic landscapes of HCCs, revealing the major pathways altered in the (phospho)proteome.

Combined protein and nucleic acid imaging reveals virus-dependent B cell and macrophage immunosuppression of tissue microenvironments.

Understanding the mechanisms of HIV tissue persistence necessitates the ability to visualize tissue microenvironments where infected cells reside; however, technological barriers limit our ability to dissect the cellular components of these HIV reservoirs. Here, we developed protein and nucleic acid in situ imaging (PANINI) to simultaneously quantify DNA, RNA, and protein levels within these tissue compartments. By coupling PANINI with multiplexed ion beam imaging (MIBI), we measured over 30 parameters simultaneously across archival lymphoid tissues from healthy or simian immunodeficiency virus (SIV)-infected nonhuman primates. PANINI enabled the spatial dissection of cellular phenotypes, functional markers, and viral events resulting from infection. SIV infection induced IL-10 expression in lymphoid B cells, which correlated with local macrophage M2 polarization. This highlights a potential viral mechanism for conditioning an immunosuppressive tissue environment for virion production. The spatial multimodal framework here can be extended to decipher tissue responses in other infectious diseases and tumor biology.

miR-579-3p Controls Hepatocellular Carcinoma Formation by Regulating the Phosphoinositide 3-Kinase-Protein Kinase B Pathway in Chronically Inflamed Liver.

Chronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development.

NUT Carcinoma: A Clinical, Morphological and Immunohistochemical Mimicker-The Role of RNA Sequencing in the Diagnostic Procedure.

Background: NUT carcinoma is a highly aggressive and rare subset of squamous cell carcinoma with grim prognosis. It is under-recognized by both pathologists and oncologists. Recognition is challenging due to its rareness and the fact that its clinical and laboratory features as well as morphological and immunohistochemical characteristics may mimic other malignancies. Case presentation: An interesting case of NUT carcinoma in a 47-year-old male with a large tumor mass in the inferior part of the mediastinum and left lung and increased levels of serum alpha fetoprotein (AFP) is described. Immunohistochemical analysis of both the primary tumor in a bronchoscopy specimen and an excisional biopsy of a subcutaneous metastasis showed positivity for AFP and leukocyte common antigen (LCA) that were misleading and resulted in diagnostic pitfalls of mediastinal germ cell tumor (clinically) and hematolymphoid neoplasm (pathologic report). Immunohistochemical demonstration of NUT protein expression revealed the proper diagnosis, which was further confirmed by RNA sequencing revealing a BRD4-NUTM1 gene fusion.Conclusions: Since NUT carcinoma can show a wide spectrum of histological and immunophenotypic features and can clinically mimic other tumors, use of RNA sequencing with identification of specific NUTM1 fusion partner could be crucial when there are discrepant clinical and histopathological findings. As well, since the category of so-called NUTM1-rearranged neoplasms is rapidly expanding, identification of NUTM1 fusion partner may be essential for the appropriate clinical management.

Vascular Damage, Thromboinflammation, Plasmablast Activation, T-Cell Dysregulation and Pathological Histiocytic Response in Pulmonary Draining Lymph Nodes of COVID-19.

Although initial immunophenotypical studies on peripheral blood and bronchoalveolar lavage samples have provided a glimpse into the immunopathology of COVID-19, analyses of pulmonary draining lymph nodes are currently scarce. 22 lethal COVID-19 cases and 28 controls were enrolled in this study. Pulmonary draining lymph nodes (mediastinal, tracheal, peribronchial) were collected at autopsy. Control lymph nodes were selected from a range of histomorphological sequelae [unremarkable histology, infectious mononucleosis, follicular hyperplasia, non-SARS related HLH, extrafollicular plasmablast activation, non-SARS related diffuse alveolar damage (DAD), pneumonia]. Samples were mounted on a tissue microarray and underwent immunohistochemical staining for a selection of immunological markers and in-situ hybridization for Epstein Barr Virus (EBV) and SARS-CoV-2. Gene expression profiling was performed using the HTG EdgeSeq Immune Response Panel. Characteristic patterns of a dysregulated immune response were detected in COVID-19: 1. An accumulation of extrafollicular plasmablasts with a relative paucity or depletion of germinal centers. 2. Evidence of T-cell dysregulation demonstrated by immunohistochemical paucity of FOXP3+, Tbet+ and LEF1+ positive T-cells and a downregulation of key genes responsible for T-cell crosstalk, maturation and migration as well as a reactivation of herpes viruses in 6 COVID-19 lymph nodes (EBV, HSV). 3. Macrophage activation by a M2-polarized, CD163+ phenotype and increased incidence of hemophagocytic activity. 4. Microvascular dysfunction, evidenced by an upregulation of hemostatic (CD36, PROCR, VWF) and proangiogenic (FLT1, TEK) genes and an increase of fibrin microthrombi and CD105+ microvessels. Taken together, these findings imply widespread dysregulation of both innate and adoptive pathways with concordant microvascular dysfunction in severe COVID-19.

Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers.

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-ß1 levels between smokers and non-smokers.

Neutrophil Extracellular Traps in Fatal COVID-19-Associated Lung Injury.

An excess formation of neutrophil extracellular traps (NETs), previously shown to be strongly associated with cytokine storm and acute respiratory distress syndrome (ARDS) with prevalent endothelial dysfunction and thrombosis, has been postulated to be a central factor influencing the pathophysiology and clinical presentation of severe COVID-19. A growing number of serological and morphological evidence has added to this assumption, also in regard to potential treatment options. In this study, we used immunohistochemistry and histochemistry to trace NETs and their molecular markers in autopsy lung tissue from seven COVID-19 patients. Quantification of key immunomorphological features enabled comparison with non-COVID-19 diffuse alveolar damage. Our results strengthen and extend recent findings, confirming that NETs are abundantly present in seriously damaged COVID-19 lung tissue, especially in association with microthrombi of the alveolar capillaries. In addition, we provide evidence that low-density neutrophils (LDNs), which are especially prone to NETosis, contribute substantially to COVID-19-associated lung damage in general and vascular blockages in particular.

MIS-C-Implications for the Pediatric Surgeon: An Algorithm for Differential Diagnostic Considerations.

BACKGROUND: multisystem inflammatory syndrome in children (MIS-C) is a new disease associated with a recent infection with severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). Affected children can present predominantly with abdominal symptoms, fever and high inflammatory parameters that might lead to a consult by the pediatric surgeon and an indication for surgery. METHODS: clinical data of three patients with MIS-C that underwent surgery were collected. Histopathological analysis of the appendix was performed. RESULTS: we present the clinical course of three children with fever, abdominal pain and vomiting for several days. Clinical examination and highly elevated inflammation markers led to indication for laparoscopy; appendectomy was performed in two patients. Because of intraoperative findings or due to lack of postoperative improvement, all patients were reevaluated and tested positive for MIS-C associated laboratory parameters and were subsequently treated with corticosteroids, intravenous immunoglobulins, acetyl salicylic acid and/or light molecular weight heparin. CONCLUSIONS: we discuss the implications of MIS-C as a new differential diagnosis and stress the importance of assessing the previous medical history, identifying patterns of symptoms and critically surveilling the clinical course. We implemented an algorithm for pediatric surgeons to consider MIS-C as a differential diagnosis for acute abdomen that can be integrated into the surgical workflow.