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The ongoing coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has triggered a devastating global health, social and economic crisis. The RNA nature and broad circulation of this virus facilitate the accumulation of mutations, leading to the continuous emergence of variants of concern with increased transmissibility or pathogenicity (1) . This poses a major challenge to the effectiveness of current vaccines and therapeutic antibodies (1, 2) . Thus, there is an urgent need for effective therapeutic and preventive measures with a broad spectrum of action, especially against variants with an unparalleled number of mutations such as the recently emerged Omicron variant, which is rapidly spreading across the globe (3) . Here, we used combinatorial antibody phage-display libraries from convalescent COVID-19 patients to generate monoclonal antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein with ultrapotent neutralizing activity. One such antibody, NE12, neutralizes an early isolate, the WA-1 strain, as well as the Alpha and Delta variants with half-maximal inhibitory concentrations at picomolar level. A second antibody, NA8, has an unusual breadth of neutralization, with picomolar activity against both the Beta and Omicron variants. The prophylactic and therapeutic efficacy of NE12 and NA8 was confirmed in preclinical studies in the golden Syrian hamster model. Analysis by cryo-EM illustrated the structural basis for the neutralization properties of NE12 and NA8. Potent and broadly neutralizing antibodies against conserved regions of the SARS-CoV-2 spike protein may play a key role against future variants of concern that evade immune control.

Older age at the time of infection with hepatitis viruses is associated with an increased risk of liver fibrosis progression. We hypothesized that the pace of fibrosis progression may reflect changes in gene expression within the aging liver. We compared gene expression in liver specimens from 54 adult donors without evidence of fibrosis, including 36 over 40 y old and 18 between 18 and 40 y old. Chitinase 3-like 1 (CHI3L1), which encodes chitinase-like protein YKL-40/CHI3L1, was identified as the gene with the greatest age-dependent increase in expression in liver tissue. We investigated the cellular source of CHI3L1 in the liver and its function using liver tissue specimens and in vitro models. CHI3L1 expression was significantly higher in livers of patients with cirrhosis of diverse etiologies compared with controls represented by patients who underwent liver resection for hemangioma. The highest intrahepatic CHI3L1 expression was observed in cirrhosis due to hepatitis D virus, followed by hepatitis C virus, hepatitis B virus, and alcohol-induced cirrhosis. In situ hybridization of CHI3L1 messenger RNA (mRNA) identified hepatocytes as the major producers of CHI3L1 in normal liver and in cirrhotic tissue, wherein hepatocytes adjacent to fibrous septa showed higher CHI3L1 expression than did those in more distal areas. In vitro studies showed that recombinant CHI3L1 promotes proliferation and activation of primary human hepatic stellate cells (HSCs), the major drivers of liver fibrosis. These findings collectively demonstrate that CHI3L1 promotes liver fibrogenesis through a direct effect on HSCs and support a role for CHI3L1 in the increased susceptibility of aging livers to fibrosis progression.

Anna Pomerenke,1 Simon R Lea,1 Sarah Herrick,2 Mark A Lindsay,3 Dave Singh1 1Centre for Respiratory Medicine and Allergy, Institute of Inflammation and Repair, Manchester Academic Health Science Centre, The University of Manchester and University Hospital of South Manchester, NHS Foundation Trust, 2Institute of Inflammation and Repair, Manchester Academic Health Science Centre, University of Manchester, Manchester, 3Department of Pharmacy and Pharmacology, University of Bath, Bath, UK Purpose: Viruses are a common cause of exacerbations in chronic obstructive pulmonary disease (COPD). They activate toll-like receptors (TLRs) 3, 7, and 8, leading to a pro-inflammatory response. We have characterized the responses of TLR3 and TLR7/8 in lung tissue explants from COPD patients and control smokers.Methods: We prepared lung whole tissue explants (WTEs) from patients undergoing surgery for confirmed or suspected lung cancer. In order to mimic the conditions of viral infection, we used poly(I:C) for TLR3 stimulation and R848 for TLR7/8 stimulation. These TLR ligands were used alone and in combination. The effects of tumor necrosis factor α (TNFα) neutralization and dexamethasone on TLR responses were examined. Inflammatory cytokine release was measured by enzyme-linked immunosorbent assay and gene expression by quantitative real-time polymerase chain reaction.Results: WTEs from COPD patients released higher levels of pro-inflammatory cytokines compared with WTEs from smokers. Activation of multiple TLRs led to a greater than additive release of TNFα and CCL5. TNFα neutralization and dexamethasone treatment decreased cytokine release.Conclusion: This WTE model shows an enhanced response of COPD compared with controls, suggesting an increased response to viral infection. There was amplification of innate immune responses with multiple TLR stimulation. Keywords: COPD, poly(I:C), R848, cytokines, lung explant

Human lung cancer is one of the leading causes of death worldwide, with nearly 2 million of new cases diagnosed each year, often too late for a successful therapeutic intervention. In this chapter, organotypic models of lung cancer will be reviewed. Ex vivo tissue explants, spheroids, organoids, and novel bioengineering approaches are currently being used to study human lung cancer. Although there is no ideal method that will fully recapitulate the complex human lung architecture, the three-dimensional (3D) organotypic models described here represent a major advance from classical two-dimensional (2D) tissue culture models. Organotypic tissue cultures are better at predicting in vivo tissue responses to anticancer drugs or carcinogenic toxins. In addition, there is also a possibility to use these systems as part of personalized medicine (e.g., to assist clinicians in selecting appropriate therapeutic choices based on individual patient-derived tissue responses to drugs in vitro). There is still a need for improvement in the 3D culture systems, such as automation for high-throughput testing and reduction of costs. However, given the large number of failed clinical trials due to safety or efficacy reasons, more attention should be given to these more physiologically relevant 3D organotypic tissue culture models.

Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN). Despite expression of all known HCV-entry factors, in vitro models based on hepatoma cell lines do not fully reproduce the in vivo susceptibility of liver cells to primary HCV isolates, implying the existence of additional host factors which are critical for HCV entry and/or replication. Likewise, HCV replication is severely impaired within hepatocellular carcinoma (HCC) tissue in vivo, but the mechanisms responsible for this restriction are presently unknown. Here, we identify tumor-associated calcium signal transducer 2 (TACSTD2), one of the most downregulated genes in primary HCC tissue, as a host factor that interacts with CLDN1 and OCLN and regulates their cellular localization. TACSTD2 gene silencing disrupts the typical linear distribution of CLDN1 and OCLN along the cellular membrane in both hepatoma cells and primary human hepatocytes, recapitulating the pattern observed in vivo in primary HCC tissue. Mechanistic studies suggest that TACSTD2 is involved in the phosphorylation of CLDN1 and OCLN, which is required for their proper cellular localization. Silencing of TACSTD2 dramatically inhibits HCV infection with a pan-genotype effect that occurs at the level of viral entry. Our study identifies TACSTD2 as a novel regulator of two major HCV-entry factors, CLDN1 and OCLN, which is strongly downregulated in malignant hepatocytes. These results provide new insights into the complex process of HCV entry into hepatocytes and may assist in the development of more efficient cellular systems for HCV propagation in vitro., Author summary Infection with hepatitis C virus (HCV) is associated with a high risk of developing hepatocellular carcinoma (HCC). However, it is unknown whether HCV promotes HCC indirectly, through chronic inflammation, fibrosis and liver regeneration, or directly, in a manner similar to other oncogenic viruses. We recently demonstrated that HCV replication within HCC tissue is severely hampered, and the present study identifies a potential mechanism for this restriction. We found that HCV RNA suppression in the tumor correlates with disruption of the typical linear distribution of two major HCV-entry cofactors, claudin-1 (CLDN1) and occludin (OCLN), which mediate viral entry into hepatocytes. This abnormal pattern was associated with downregulation of tumor-associated calcium signal transducer 2 (TACSTD2), an essential gene for maintaining the proper cellular localization of CLDN1 and OCLN. Thus, this study identifies TACSTD2 as a novel host factor that regulates the cellular localization of two major HCV-entry cofactors. A major highlight of our study is that our in vitro observations recapitulate the in vivo findings in patients with HCC, corroborating their biological relevance. These results shed new light on the complex process of HCV entry and may have relevance for the pathogenesis, treatment and prevention of HCV infection.

Human lung cancer is one of the leading causes of death worldwide, with nearly 2 million of new cases diagnosed each year, often too late for a successful therapeutic intervention. In this chapter, organotypic models of lung cancer will be reviewed. Ex vivo tissue explants, spheroids, organoids, and novel bioengineering approaches are currently being used to study human lung cancer. Although there is no ideal method that will fully recapitulate the complex human lung architecture, the three-dimensional (3D) organotypic models described here represent a major advance from classical two-dimensional (2D) tissue culture models. Organotypic tissue cultures are better at predicting in vivo tissue responses to anticancer drugs or carcinogenic toxins. In addition, there is also a possibility to use these systems as part of personalized medicine (e.g., to assist clinicians in selecting appropriate therapeutic choices based on individual patient-derived tissue responses to drugs in vitro). There is still a need for improvement in the 3D culture systems, such as automation for high-throughput testing and reduction of costs. However, given the large number of failed clinical trials due to safety or efficacy reasons, more attention should be given to these more physiologically relevant 3D organotypic tissue culture models.