A novel mouse model of COVID-19

OBJECTIVES/GOALS: Rodents are the most widely used experimental animals to study disease mechanisms due to their availability and cost-effectiveness. An international drive to investigate the pathophysiology of COVID-19 is inhibited by the resistance of rats and mice to SARS-CoV-2 infection. Our goal was to establish an appropriate small animal model. METHODS/STUDY POPULATION: To recreate the cytokine storm that is associated with COVID-19, we injected angiotensin converting enzyme 2 knockout (ACE2KO) mice (C57BI/6 strain) with lipopolysaccharide (LPS) intraperitoneally and measured the expression of multiple cytokines as a function of time and LPS dose. We then chose a minimum dose (500ug/kg) and time (3h) when multiple cytokines were elevated to measure lung injury scores using a point-counting technique on tissue sections stained with hematoxylin and eosin. The data are expressed as mean percentage of grid points lying within the peribronchial and superficial area in up to 20 fields. Percentage of peribronchial and superficial intrapulmonary hemorrhage, congestion, neutrophil infiltration and area of alveolar space were all assessed. RESULTS/ANTICIPATED RESULTS: Compared to the wildtype group (WT-G), the LPS-injected ACE2KO mice (LPS-G) exhibited a higher percentage of peribronchial intrapulmonary hemorrhage [(%): LPS-G, 10.56 ± 2.06 vs. WT-G, 5.59 ± 0.53;p DISCUSSION/SIGNIFICANCE: Establishing this novel mouse model of COVID-19 will facilitate studies investigating tissue-specific mechanisms of pathogenesis in this disease. This model can also be used to discover novel therapeutic targets and the design of clinical trials focusing on diagnostics, treatments and outcomes in COVID-19.

Lipid Polysaccharides have a Detrimental Effect on the Function of the Ovaries and Uterus in Mice through Increased Pro-Inflammatory Cytokines.

As the number of coronavirus disease 2019 (COVID-19) vaccinations increases, various side effects are being reported, and menstrual abnormalities have been reported as a side effect in women. However, it is still unclear whether the COVID-19 vaccine has detrimental effects on the female reproductive system. Therefore, we investigated the effect of excessive immune response on reproductive function by administering Lipopolysaccharides (LPS) instead of the COVID-19 vaccine. The immune response in mice was induced by injection of LPS. Mice injected with saline 5 times were used as a control group, and mice injected with LPS 5 times were used as an experimental group. Repeated administration of LPS significantly reduced the number of corpus luteum (CL). On the other hand, the injection of LPS did not affect the development of follicles leading before the CL. The expression of the apoptosis-related genes Fas and Fas-L increased in the experimental group. In addition, the expression of the inflammation-related genes increased in the experimental group. In this study, we confirmed that LPS had detrimental effects on the uterus and ovaries in mice. These results suggest that injection of LPS can cause immune reactions within the uterus and ovaries and cause hormonal changes, which can have adverse effects such as abnormal operation or bleeding of the menstrual cycle. These results are expected to help determine the cause of decreased reproductive function, infertility, or physiological disorders caused by the COVID-19 vaccine.

Introduction pages, Notulae Scientia Biologicae 14(3), 2022

Notulae Scientia Biologicae (http://www.notulaebiologicae.ro), Issue 3, Volume 14, 2022: The papers published in this issue represent interesting novelties in different topics of life science. Among the exciting research or reviews, we invite readers to find news about: Micropropagation and potential of bioactive compounds of saffron (Crocus sativus L.) for nutrition and health;Anatomical, physiological, biochemical and molecular responses of Eucalyptus spp. under water deficit conditions and characteristics of Tunisian arid species;Escherichia coli infection, a negative prognostic factor on the evolution of patients with surgical diseases;Biological characteristics and mortality in patients with diabetes and COVID-19;The influence of Staphylococcus infections on the evolution of hospitalized patients: The experience of the surgical department of IRGH Cluj-Napoca;Parquetina nigrescens leaf infusion: a food-based approach for the management of diet-induced iron deficiency in weanling rats;Evaluation of the effects of calabash chalk on the haematological profile of Wistar rats;Inhibitory potential of rutin on lipopolysaccharide-induced toxicity and inflammatory response of raw U937 cells and macrophages;Hypoglycemic and in vitro antioxidant activities of Stereospermum kunthianum stem bark hydromethanol extract;Polyploidization and speciation: patterns of natural hybridization and gene flow in basil (Ocimum spp.);Increasing liana biomass and carbon stocks in tropical dry evergreen forests of southern India.

Structural characterization and anti-inflammatory activity of fucoidan isolated from Ecklonia maxima stipe

Enzyme-assisted hydrolysis is frequendy used as a cost-effective and efficient method to obtain functional ingredients from bioresources. This study involved die enzyme-assisted hydrolyzation and purification of fucoidan from Ecklonia maxima stipe and die investigation of its anti-inflammatory activity in lipopolysaccharide (LPS)-induced RAW 264.7 cells. Fucoidans of Viscozyme-assisted hydrolysate from E. maxima (EMSFs) harvested in Jeju, Korea. Structural and chemical characterizations were performed using fourier transform infrared spectroscopy, scanning electron microscope, and monosaccharide analysis. Among fucoidans, EMSF6 was rich in fucose and sulfate and had a similar structural character to commercial fucoidan. EMSF6 showed a strong inhibitory effect on nitric oxide generation in LPS-induced RAW 264.7 cells and significantly decreased die production of LPS-induced pro-inflammatory cytokines, including interleukin-6, interleukin-1 p, and tumor necrosis factor a. The anti-inflammatory potential of EMSF6 was mediated through the down-regulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Thus, fucoidans from&temppound;. maxima stipe are promising candidates for functional food products.

Differential Regulation of CD45 Expression on Granulocytes, Lymphocytes, and Monocytes in COVID-19.

CD45 is a transmembrane glycoprotein and protein tyrosine phosphatase expressed on the surface of all nucleated hematopoietic cells. While there is increasing evidence demonstrating the involvement of CD45 in immune system regulation, no information on CD45 expression in inflammation and sepsis is currently available. Therefore, we determined the CD45 surface expression on granulocytes, lymphocytes, and monocytes in patients with COVID-19 and healthy volunteers in both absence and presence of lipopolysaccharide (LPS). Following approval by the local ethics committee, whole blood samples were obtained from patients with COVID-19 infection on day 1 of hospital admission and healthy volunteers. Samples were incubated in absence and presence of LPS and CD45 was measured in granulocytes, lymphocytes, and monocytes using flow cytometry. In comparison with healthy individuals, COVID-19 patients showed an increased CD45 expression on the surface of granulocytes (+35%, p < 0.02) and lymphocytes (+39%, p < 0.0001), but a reduced CD45 expression on monocytes (-35%, p < 0.0001). LPS incubation of whole blood from healthy individuals increased the CD45 expression on granulocytes (+430%, p < 0.0001), lymphocytes (+32%, p = 0.0012), and monocytes (+36%, p = 0.0005), respectively. LPS incubation of whole blood samples from COVID-19 patients increased the CD45 expression on granulocytes and monocytes, and decreased the CD45 expression on lymphocytes. In conclusion, CD45 expression on leucocytes is altered: (1) in COVID-19 patients, and (2) in in vitro endotoxemia in a complex cell-specific way, thus representing a new immunoregulatory mechanism.

The First Optimization Process from Cultivation to Flavonoid-Rich Extract from Moringa oleifera Lam. Leaves in Brazil

Flavonoids are significant antioxidant and anti-inflammatory agents and have multiple potential health applications. Moringa oleifera is globally recognized for its nutritional and pharmacological properties, correlated to the high flavonoid content in its leaves. However, the bioactive compounds found in plants may vary according to the cultivation, origin, season, and extraction process used, making it difficult to extract reliable raw material. Hence, this study aimed to standardize the best cultivation and harvest season in Brazil and the best extraction process conditions to obtain a flavonoid-rich extract from M. oleifera as a final product. Firstly, ultrasound-assisted extraction (UAE) was optimized to reach the highest flavonoid content by three-level factorial planning and response surface methodology (RSM). The optimal cultivation condition was mineral soil fertilizer in the drought season, and the optimized extraction was with 80% ethanol and 13.4 min of extraction time. The flavonoid-rich extract was safe and significantly decreased reactive oxygen species (ROS) and nitric oxide (NO) in LPS-treated RAW 264.7 cells. Lastly, the major flavonoids characterized by HPLC-ESI-QTRAP-MS/MS were compounds derived from apigenin, quercetin, and kaempferol glycosides. The results confirmed that it was possible to standardize the flavonoid-rich extract leading to a standardized and reliable raw material extracted from M. oleifera leaves.

Brain pathophysiology in SARS-CoV-2 disease

OBJECTIVES/GOALS: The SARS-CoV-2 (Severe Acute Respiratory Syndrome CoronaVirus-2), which underlies the current COVID-19 pandemic, among other tissues, also targets the central nervous system (CNS). The goal of this study is to investigate mechanisms of neuroinflammation in Lipopolysaccharides (LPS)-treated mouse model and SARS-CoV-2-infected hamsters. METHODS/STUDY POPULATION: In this research I will assay vascular reactivity of cerebral vessels to assess vascular dysfunction within the microcirculation. I will determine expression of proinflammatory cytokines, coagulation factors and AT1 receptors (AT1R) in isolated microvessels from the circle of Willis to assess inflammation, thrombosis and RAS activity in the microvasculature. LPS and SARS-CoV-2, are both associated with coagulopathies and because of that I will measure concentration of PAI-1, von Willebrand Factor, thrombin and D-dimer to assess the thrombotic pathway in the circulation. Histology and immunohistochemistry will assess immune cell type infiltration into the brain parenchyma, microglia activation and severity of neuroinflammation and neural injury. RESULTS/ANTICIPATED RESULTS: We hypothesize that under conditions of reduced ACE2 (e.g., SARS-CoV-2 infection), AT1R activity is upregulated in the microvasculature. In the presence of an inflammatory insult, these AT1Rs promote endothelialitis and immunothrombosis through pro-thrombotic pathways and pro-inflammatory cytokine production leading to endothelial dysfunction in the microvasculature, blood brain barrier (BBB) injury, deficits in cognition and increased anxiety. We will test this hypothesis through 2 aims: Aim 1: Determine the role of the pro-injury arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. Aim 1: Determine the role of the protective arm of the RAS in the pathophysiology of the brain in animal models of neuroinflammation and COVID-19. DISCUSSION/SIGNIFICANCE: This study will provide insights that will complement on-going clinical trials on angiotensin type 1 receptor (AT1R) blockers (ARBs) in COVID-19. This research is a necessary first step in understanding mechanisms of brain pathogenesis that can set the groundwork for future studies of more complex models of disease.

Countermeasures against COVID-19 from the viewpoints of dental diseases: strengthening innate immunity with the toothpick method of brushing. (Special Issue: COVID-19 and historical pandemics part 2.)

Coronavirus disease (COVID-19) patients with periodontal disease have an 8.8-fold higher mortality rate than those in the patients without periodontal diseases. This was higher than the odds ratio for patients with diabetes. Periodontal disease is associated with ulcers in the periodontal pocket, and gram-negative bacteria called periodontal pathogens invade the tissue through ulcers. Bacteria in the ulcer site are phagocytosed and sterilized by leukocytes. Following the autolysis of leukocytes, lipopolysaccharides (LPS) on the bacterial cell wall spread throughout the body, which is a major cause of multiple organ failure. Thus, periodontal disease is considered to contribute to the mortality rate of COVID-19. Ulcers in the periodontal pocket can be repaired using by a new developed brushing method called the toothpick method. The toothpick method can significantly improve gingival bleeding in one week, which is quicker than conventional periodontal treatment methods. Mechanical stimulation by the toothbrush causes gingival basal cells, fibroblasts, vascular endothelial cells and osteoblasts to proliferate and repair the tissue. However, these cell proliferations cease to occur 0.5 mm away from where the toothbrush bristles make contact with the gingiva. The toothpick method of brushing is characterized by its ability to stimulate the interdental gingiva, which is the initial site of periodontitis. As the toothpick method can repair periodontal ulcers, it will strengthen biological defense mechanisms against chronic degenerative and infectious diseases.

Post-treatment with propofol inhibits inflammatory response in LPS-induced alveolar type II epithelial cells.

Over-inflammation and severe lung injury are major causes of morbidity and mortality in patients with coronavirus disease 2019 (COVID-19). With the COVID-19 pandemic, an increasing number of patients with preexisting lung injury and inflammation are undergoing surgery or artificial ventilation under sedation in intensive care units, where 2,6-diisopropylphenol (propofol) is a commonly used drug for sedation. The aim of the present study was to investigate whether post-inflammation treatment with propofol protects epithelial type II cells against inflammation in an in vitro model of inflammation. The A549 cell line, characterised as epithelial type II cells, were exposed to lipopolysaccharide (LPS) for 2 h and subsequently treated with different concentrations of propofol (0, 10, 25 or 50 µM) for 3 h. Western blot and reverse transcription-quantitative PCR analyses were used to detect the protein and mRNA expression levels, respectively, of CD14 and Toll-like receptor 4 (TLR4). Immunofluorescence staining was used to detect the in situ CD14 and TLR4 expression in epithelial type II cells. Tumor necrosis factor (TNF)-α production was also examined using ELISA. LPS significantly increased the expression of CD14 and TLR4, as well as the secretion of TNF-α. Post-treatment with 25 and 50 µM propofol of the LPS-treated cells significantly decreased CD14 and TLR4 expression, as well as TNF-α secretion, compared with the cells treated with LPS only, indicating that post-treatment with propofol alleviated inflammation and this effect was dose-dependent. The present study suggested that treatment with propofol after LPS administration has a protective effect on epithelial type II cells.

Mechanism of HanShi ZuFei formula in treatment of COVID-19 based on network pharmacology and molecular docking technology

Objective: To explore the mechanism of Hanshi Zufei Formula (HSZFF) treating coronavirus disease 2019 (COVID-19) by the data mining analysis of network pharmacology and the molecular docking.

Mechanism of Xuanbai Qingfei Jiedu decoction in the treatment of COVID-19 based on network pharmacology

Objective: To explore the pharmacological mechanism of Xuanbai Qingfei Jiedu Decoction in the treatment of coronavirus disease 2019 (COVID-19) on account of network pharmacology.

Urban air PM modifies differently immune defense responses against bacterial and viral infections in vitro.

Epidemiological evidence has shown the association between exposure to ambient fine particulate matter (PM) and increased susceptibility to bacterial and viral respiratory infections. However, to date, the underlying mechanisms of immunomodulatory effects of PM remain unclear. Our objective was to explore how exposure to relatively low doses of urban air PM alters innate responses to bacterial and viral stimuli in vitro. We used secondary alveolar epithelial cell line along with monocyte-derived macrophages to replicate innate lung barrier in vitro. Co-cultured cells were first exposed for 24 h to PM2.5-1 (particle aerodynamic diameter between 1 and 2.5 µm) and subsequently for an additional 24 h to lipopolysaccharide (TLR4), polyinosinic-polycytidylic acid (TLR3), and synthetic single-stranded RNA oligoribonucleotides (TLR7/8) to mimic bacterial or viral stimulation. Toxicological endpoints included pro-inflammatory cytokines (IL-8, IL-6, and TNF-α), cellular metabolic activity, and cell cycle phase distribution. We show that cells exposed to PM2.5-1 produced higher levels of pro-inflammatory cytokines following stimulation with bacterial TLR4 ligand than cells exposed to PM2.5-1 or bacterial ligand alone. On the contrary, PM2.5-1 exposure reduced pro-inflammatory responses to viral ligands TLR3 and TLR7/8. Cell cycle analysis indicated that viral ligands induced cell cycle arrest at the G2-M phase. In PM-primed co-cultures, however, they failed to induce the G2-M phase arrest. Contrarily, bacterial stimulation caused a slight increase in cells in the sub-G1 phase but in PM2.5-1 primed co-cultures the effect of bacterial stimulation was masked by PM2.5-1. These findings indicate that PM2.5-1 may alter responses of immune defense differently against bacterial and viral infections. Further studies are required to explain the mechanism of immune modulation caused by PM in altering the susceptibility to respiratory infections.

EphrinA1/EphA2 Promotes Epithelial Hyperpermeability Involving in Lipopolysaccharide-induced Intestinal Barrier Dysfunction.

Background/Aims: Lipopolysaccharide (LPS) is the key factor inducing mucosal and systemic inflammation in various intestinal and parenteral diseases, which could initially disrupt the epithelial barrier function. EphrinA1/ephA2 is speculated to increase the epithelial permeability for its "repulsive interaction" between adjacent cells. This study aim to investigate the role of ephrinA1/ephA2 in LPS-induced epithelial hyperpermeability. Methods: In vivo model challenged with oral LPS in C57BL/6 mice and in vitro model exposed to LPS in Caco2 monolayer were established. The barrier function was assessed including expression of tight junction proteins (occludin and claudin-1), transepithelial electrical resistance, and permeability to macromolecules (fluorescein isothiocyanate-labeled fluorescent dextran 4 kDa [FD4]). Moreover, the expression and phosphorylation of ephrinA1/ephA2 were quantified, and its roles in the process of epithelial barrier disruption were confirmed via stimulating ephA2 with ephrinA1-Fc chimera (ephrinA1-Fc) and inactivating ephA2 with ephA2-Fc chimera (ephA2-Fc), or ephA2 monoclonal antibody (ephA2-mab), as well as inhibiting extracellular signal-regulated kinase 1/2 (ERK1/2) with PD98059. Results: LPS induced significant barrier dysfunction with dismissed occludin and claudin-1 expression, reduced transepithelial electrical resistance and increased FD4 permeability, accompanied by upregulated ephrinA1/ephA2 pathway and phosphorylation of ephA2 receptor. Furthermore, ephA2-Fc, and ephA2-mab ameliorated LPS-induced epithelial hyperpermeability, which was also inhibited by PD98059. Additionally, ephrinA1-Fc led to apparent epithelial leakage in Caco2 monolayer by promoting the phosphorylation of ERK1/2, which could be obviously blocked by ephA2-mab and PD98059. Conclusion: EphrinA1/ephA2 promotes epithelial hyperpermeability with an ERK1/2-dependent pathway, which involves in LPS-induced intestinal barrier dysfunction.