IDDF2023-ABS-0045 Spatiotemporal immune landscape of Mas-driven pro-inflammatory microenvironment with hyperactivated glycolysis in acetaminophen-induced liver injury

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A standardized procedure for quantitative evaluation of residual viral activity on antiviral treated textiles

The SARS-CoV-2 pandemic has increased the demand for antiviral technologies to mitigate or prevent the risk of viral transmission. Antiviral treated textiles have the potential to save lives, especially in healthcare settings that rely on reusable patient-care textiles and personal protective equipment. Currently, little is known about the role of textiles in cross-contamination and pathogen transmission, despite the wealth of information on hard surfaces and fomites harboring viruses that remain viable in certain circumstances. In addition, there is no international standard method for evaluating residual viral activity on textiles, which would allow a thorough investigation of the efficacy of antiviral textile products. Therefore, this pilot study aims to develop and refine a standardized protocol to quantitatively evaluate residual viral activity on antiviral textiles. Specifically, we focused on general textiles, such as bed linens, commonly used in healthcare settings for patient care. The Tissue Culture Infectious Dose 50 (TCID50) method is frequently used to quantitatively evaluate viral infectivity on textiles, but has not been established as a standard. This procedure involves observing the cytopathic effect of a given virus on cells grown in a 96-well plate after several days of incubation to determine the infectivity titer. We used HCoV-229E and Huh-7 human liver cancer cells for this investigation. We worked to improve the TCID50 method through variations of different steps within the protocol to attain reproducible results. Our proposed optimized hybrid protocol has shown evidence that the protocol is technically simpler and more efficient, and provides successful, consistent results. The analysis showed a significant difference between the treated fabric compared with controls.

Hepatocyte-Directed Delivery of Lipid-Encapsulated Small Interfering RNA.

Lipid formulations for cell transfection are among the most efficient systems for nucleic acid delivery. During the COVID-19 pandemic, lipid-encapsulated RNA (lipid nanoparticles, LNP) has succeeded as a superior vaccine. Moreover, other similar lipid nanocarriers for siRNA are approved and many are on the pipelines. While lipid encapsulation required several devices for the mixing of components, lipoplex technology allows to rapidly mix nucleic acids and positively charged lipids for cell transfection. In vivo, hepatocytes are important target cells of lipid formulated RNAi. This chapter describes the state-of-the-art lipoplex and LPN manufacturing for treating primary hepatocytes with lipid formulations. Furthermore, protocols for isolating murine hepatocytes and for transfecting these cells with pharmaceutically relevant lipid formulations are provided and discussed.

Tocilizumab Is Effective in Reducing Inflammation in T2D–COVID-Organ-on-a-Chip Model

The COVID-pandemic has contributed to more than 5 million deaths worldwide in the last two years. Co-morbid conditions such as Type 2 Diabetes (T2D) , HTN, obesity, and CKD have been associated with increased mortality with COVID-19. In a large meta-analysis, the relative risk of mortality was 1.54 for patients with T2D and COVID-19. Thus, there is an imperative need to develop a platform for rapid and reliable drug screening/selection against COVID-related morbidity/mortality in T2D patients. With limited translatability of in vitro and small animal models to humans, human organ-on-a-chip models are an attractive platform to model in vivo disease conditions and test potential therapeutics. We seeded T2D or nondiabetes patient-derived macrophage and human liver sinusoidal endothelial cells along with normal hepatocytes and kupffer cells in the liver-on-a-chip (LAMPS - Liver Acinus MicroPhysiological System) developed by our group, perfused with media mimicking normal fasting or late metabolic syndrome (LMS - high levels of glucose, fatty acids, insulin, glucagon) states. We transduced both macrophage and endothelial cells to overexpress the SARS-CoV2-S (spike) protein and compared it with a control lentivirus transduction. We found that T2D cells overexpressing S-protein in LMS media (T2D chip) displayed an increased secretion of inflammatory cytokines compared to the nondiabetes chip over days. We then tested the effect of Tocilizumab (IL6-receptor antagonist) in T2D chips. Compared to vehicle control, Tocilizumab significantly decreased the S-protein induced inflammatory cytokine secretion in T2D chips but not in nondiabetes chips, indicating its higher efficacy in severe disease states only. This is consistent with what was observed in large clinical trials providing confirmatory evidence that the LAMPS T2D and nondiabetes chips serve as a relevant in vitro model system to replicate human in vivo pathophysiology of COVID and for screening potential therapeutics.

Marine Microbial Diversity as Source of Bioactive Compounds

Four new cytotoxic indole-diterpenoids (penerpenes K-N), along with twelve other known compounds, have been discovered by Dai et al. from the fermentation broth produced by adding L-tryptophan to the culture medium of Penicillium sp. Three compounds (penerpene N, epipaxilline, emindole SB) were found to be cytotoxic to cancer cell lines, of which the known compound, epipaxilline, was the most active and showed cytotoxic activity against the human liver cancer cell line BeL-7402 with an IC50 value of 5.3 μM. Moreover, six compounds, namely paxilline, 7-hydroxyl-13-dehydroxypaxilline, 7-hydroxypaxilline-13-ene, 4a-demethylpaspaline-4a-carboxylic acid, PC-M6 and emindole SB, showed antibacterial activities against Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633 [3]. [...]the authors of this manuscript have also reported, for the first time, a putative lysosomal acid lipase produced by a green microalgae [7]. In this review, the authors have summarized the list of 145 natural products isolated from microorganisms associated with sea cucumbers between 2000 and 2021, which include polyketides, alkaloids and terpenoids as well as their reported biological activities [8].

Molecular detection of Avian coronavirus (infectious bronchitis virus) associated with poultry enteritis

Poultry enteritis is an important multifactorial disease. Avian coronavirus (ACV) is one of many viruses related to enteric diseases and infectious bronchitis. Aim of this study was to find out the occurrence of ACV in enteritis affected broiler, molecular detection, phylogenetic analysis of avian corona virus and to examine intestine and liver for gross and microscopic lesions. Dead poultry birds (N=604) affected with enteritis were examined for presence of ACV. Intestinal samples of four birds were pooled to make one biological sample enteric ACV as the causative agent of enteritis in commercial poultry sector in and around four major districts of Rajasthan by RT-PCR. Molecular characterization was carried out by partial gene sequencing. Liver and intestine were examined grossly during post-mortem and by histopathology. Out of 151 pooled samples tested 51 (35.10%) were found positive for ACV. Prevalence of enteric ACV was highest in Ajmer (45.94%) and lowest in Dungarpur (23.07%) districts. 0-1 weeks age chicken flocks were found more susceptible for enteric ACV with 33.80% prevalence. Comparison of ACV sequence of this study revealed nucleotide (nt) identities from 99.44% among themselves, 99.44% with ACV from abroad. The amino acid (aa) identities of ACV of this study among themselves and with abroad sequences was 47.06 to 100%. Further severe congestion in intestine and necrotic patches on liver were recorded. Histopathology showed severe villous atrophy, congestion and cystic glands in sub-mucosa in intestine and severe congestion and haemorrhages along with infiltration of inflammatory cells in liver parenchyma.

Interspecies Comparisons of the Effects of Potential Antiviral 3-Amidinophenylalanine Derivatives on Cytochrome P450 1A2 Isoenzyme.

In vitro models of animals vulnerable to SARS-CoV-2 infection can support the characterization of effective antiviral drugs, such as synthetic inhibitors of the transmembrane protease serine 2 (TMPRSS2). Changes in cytochrome P450 (CYP) 1A2 activities in the presence of the potential TMPRSS2/matriptase inhibitors (MI) were measured using fluorometric and luminescent assays. Furthermore, the cytotoxicity of these inhibitors was evaluated using the MTS method. In addition, 60 min-long microsomal stability assays were performed using an UPLC-MS/MS procedure to elucidate depletion rates of the inhibitors. CYP1A2 was influenced significantly by MI-463 and MI-1900 in rat microsomes, by MI-432 and MI-482 in beagle microsomes, and by MI-432, MI-463, MI-482, and MI-1900 in cynomolgus monkey microsomes. The IC50 values in monkey microsomes were 1.30 ± 0.14 µM, 2.4 ± 1.4 µM, 0.21 ± 0.09 µM, and 1.1 ± 0.8 µM for inhibitors MI-432, MI-463, MI-482, and MI-1900, respectively. The depletion rates of the parent compounds were lower than 50%, independently of the investigated animal species. The host cell factor TMPRSS2 is of key importance for the cross-species spread of SARS-CoV-2. Studies of the in vitro biotransformation of TMPRSS2 inhibitors provide additional information for the development of new antiviral drugs.

In vitro characterization of the furin inhibitor MI-1851: Albumin binding, interaction with cytochrome P450 enzymes and cytotoxicity.

The substrate-analog furin inhibitor MI-1851 can suppress the cleavage of SARS-CoV-2 spike protein and consequently produces significant antiviral effect on infected human airway epithelial cells. In this study, the interaction of inhibitor MI-1851 was examined with human serum albumin using fluorescence spectroscopy and ultrafiltration techniques. Furthermore, the impacts of MI-1851 on human microsomal hepatic cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6 and 3A4 activities were assessed based on fluorometric assays. The inhibitory action was also examined on human recombinant CYP3A4 enzyme and on hepatocytes. In addition, microsomal stability (60 min) and cytotoxicity were tested as well. MI-1851 showed no relevant interaction with human serum albumin and was significantly depleted by human microsomes. Furthermore, it did not inhibit CYP1A2, 2C9, 2C19 and 2D6 enzymes. In human hepatocytes, CYP3A4 was significantly suppressed by MI-1851 and weak inhibition was noticed in regard to human microsomes and human recombinant CYP3A4. Finally, MI-1851 did not impair the viability and the oxidative status of primary human hepatocytes (up to 100 µM concentration). Based on these observations, furin inhibitor MI-1851 appears to be potential drug candidates in the treatment of COVID-19, due to the involvement of furin in S protein priming and thus activation of the pandemic SARS-CoV-2.

Patomorphology of the liver in pregnant with Coronavirus infection

Researchers have shown that SARS-CoV and MERS-CoV can also cause liver damage in an infected organism, but the mechanisms of injury are poorly understood. In this study, pathomorphological changes in the liver during coronavirus infection in pregnant women were studied. As material, the liver was studied at autopsy of maternal mortality from 33 coronaviruses conducted at RCPA (Republican Center for Pathological Anatomy) between 2020 and 2021. Morphological examinations of liver tissue showed that the development of various pathomorphological changes in the liver was also observed depending on the periods of coronavirus infection. In the exudative period of the coronavirus is observed a strong process of circulating in the liver, swelling, destruction and bleeding of interstitial tissue, the development of protein and hydropic dystrophy in the liver parenchyma, i.e. hepatocytes. In the second proliferative inflammatory period of the disease, there is an increase in lymphoid infiltration along the portal pathways of the liver, myxomatous metaplasia of Kupffer cells, proliferation and proliferation of fibroblasts, growth of connective tissue, portal pathways of fibrous structures, periphery and even sinusoidal wall.

COVID-19 and liver disease: Are we missing something?

Since the coronavirus disease 2019 (COVID-19) has hit the world as a pandemic, researchers all over the world have worked on its diagnostics, prognosticating factors, etc. The present study showed liver enzymes, especially aspartate aminotransferase (AST) levels, to be high in non-survivors with raised AST/alanine aminotransferase ratio. Considering the non-specific nature of AST with its presence in organs other than liver such as muscle, heart, kidney and brain makes it difficult to interpret. Even pre-existing metabolic syndrome and non-alcoholic fatty liver disease are confounding factors for deranged liver functions detected during COVID-19 disease. Therefore, the results of the study should be taken with caution.

GalNAc-siRNA conjugates: Prospective tools on the frontier of anti-viral therapeutics.

The growing use of short-interfering RNA (siRNA)-based therapeutics for viral diseases reflects the most recent innovations in anti-viral vaccines and drugs. These drugs play crucial roles in the fight against many hitherto incurable diseases, the causes, pathophysiologies, and molecular processes of which remain unknown. Targeted liver drug delivery systems are in clinical trials. The receptor-mediated endocytosis approach involving the abundant asialoglycoprotein receptors (ASGPRs) on the surfaces of liver cells show great promise. We here review N-acetylgalactosamine (GalNAc)-siRNA conjugates that treat viral diseases such as hepatitis B infection, but we also mention that novel, native conjugate-based, targeted siRNA anti-viral drugs may also cure several life-threatening diseases such as hemorrhagic cystitis, multifocal leukoencephalopathy, and severe acute respiratory syndrome caused by coronaviruses and human herpes virus.

Binding of the SARS-CoV-2 Spike Protein to the Asialoglycoprotein Receptor on Human Primary Hepatocytes and Immortalized Hepatocyte-Like Cells by Confocal Analysis.

BACKGROUND: The SARS-CoV-2 virus may have direct or indirect effects on other human organs beyond the respiratory system and including the liver, via binding of the spike protein. This study investigated the potential direct interactions with the liver by comparing the binding of SARS-CoV-2 spike proteins to human AT2-like cells, primary human hepatocytes and immortalized hepatocyte-like hybrid cells. Receptors with binding specificity for SARS-CoV-2 spike protein on AT2 cells and hepatocytes were identified. METHODS: The specific binding of biotinylated spike and spike 1 proteins to undifferentiated human E12 MLPC (E12), E12 differentiated alveolar type 2 (AT2) cells, primary human hepatocytes (PHH) and E12 human hepatocyte-like hybrid cells (HLC) was studied by confocal microscopy. We investigated the expression of ACE-2, binding of biotinylated spike protein, biotinylated spike 1 and inhibition of binding by unlabeled spike protein, two neutralizing antibodies and an antibody directed against the hepatocyte asialoglycoprotein receptor 1 (ASGr1). RESULTS: E12 MLPC did not express ACE-2 and did not bind either of spike or spike 1 proteins. AT2-like cells expressed ACE-2 and bound both spike and spike 1. Both PHH and HLC did not express ACE-2 and did not bind spike 1 protein. However, both PHH and HLC actively bound the spike protein. Biotinylated spike protein binding was inhibited by unlabeled spike but not spike 1 protein on PHH and HLC. Two commercial neutralizing antibodies blocked the binding of the spike to PHH and HLC but only one blocked binding to AT2. An antibody to the hepatocyte ASGr1 blocked the binding of the spike protein to PHH and HLC. CONCLUSION: The absence of ACE-2 receptors and inhibition of spike binding by an antibody to the ASGr1 on both PHH and HLC suggested that the spike protein interacts with the ASGr1. The differential antibody blocking of spike binding to AT2, PHH and HLC indicated that neutralizing activity of SARS-CoV-2 binding might involve additional mechanisms beyond RBD binding to ACE-2.