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Dayuanyin (DYY) has been shown to reduce lung inflammation in both coronavirus disease 2019 (COVID-19) and lung injury. This experiment was designed to investigate the efficacy and mechanism of action of DYY against hypoxic pulmonary hypertension (HPH) and to evaluate the effect of DYY on the protection of lung function. Animal welfare and experimental procedures are approved and in accordance with the provision of the Animal Ethics Committee of the Institute of Materia Medica, Chinese Academy of Medical Science. Male C57/BL6J mice were randomly divided into 4 groups: control group, model group, DYY group (800 mg.kg-1), and positive control sildenafil group (100 mg.kg-1). The animals were given control solvents or drugs by gavage three days in advance. On day 4, the animals in the model group, DYY group and sildenafil group were kept in a hypoxic chamber containing 10% +/- 0.5% oxygen, and the animals in the control group were kept in a normal environment, and the control solvent or drugs continued to be given continuously for 14 days. The right ventricular systolic pressure, right ventricular hypertrophy index, organ indices and other metrics were measured in the experimental endpoints. Meantime, the expression levels of the inflammatory factors in mice lung tissues were measured. The potential therapeutic targets of DYY on pulmonary hypertension were predicted using network pharmacology, the expression of nuclear factor kappa B (NF- kappaB) signaling pathway-related proteins were measured by Western blot assay. It was found that DYY significantly reduced the right ventricular systolic pressure, attenuated lung injury and decreased the expression of inflammatory factors in mice. It can also inhibit hypoxia-induced activation of NF- kappaB signaling pathway. DYY has a protective effect on lung function, as demonstrated by DYY has good efficacy in HPH, and preventive administration can slow down the disease progression, and its mechanism may be related to inhibit the activation of NF-kappaB and signal transducer and activator of transcription 3 (STAT3) by DYY.Copyright © 2023, Chinese Pharmaceutical Association. All rights reserved.
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The novel SARS-CoV-2 virus known to cause the COVID-19 outbreak has resulted in a global healthcare crisis that has persisted the past 3 years. Thus, understanding the mechanisms underlying this disease are vital at this time. While there are issues of research infrastructure to handle the virus and because of the refractoriness of rodents to this disease, the availability of these tools is still limited. The cytokine storm and fatality presented in patients with severe COVID-19 can be mimicked with Staphylococcal enterotoxin B (SEB)-induced Acute Respiratory Distress Syndrome (ARDS). Within ~7 days, the survival rate drops to 0% for C3H/HeJ mice exposed to a dual dose of SEB. In this study, we administered cannabidiol (CBD) intraperitoneally for 3 days pre- and post-SEB dosing and found that the clinical outcomes improved significantly. Initial evaluation of scRNASeq data from lungs comparing naive to SEB-induced ARDS mice illustrated an increase in infiltrating immune cells, and a loss in pulmonary epithelial cells in the latter group. When evaluating the effect of CBD treatment on SEB-induced ARDS, we were able to demonstrate that CBD reduced the macrophage population. To characterize the mechanism by which CBD treatment ameliorated the inflammatory response, we found that CBD treated mice had significant reduction in infiltrating immune cells and alveolar thickening. This same histology and infiltration is presented in ARDS. MicroRNA expression analysis showed a significant increase in the expression mmu-miR-298-5p and mmu-miR- 566 with CBD treatment. Ingenuity Pathway Analysis (IPA) indicated that the dysregulated miRNAs were also implicated in pathways associated with macrophage activation, respiratory disease and inflammation, interferon stimulated genes, as well as genes which have been upregulated in the disease state of this model. These targets include but are not limited to Cebpb, Efhd2, Stat3, Socs3, Cxcl5, Gbp2, and Birc3. This finding offers insights for the development of preventive and therapeutic strategies in the treatment of ARDS, including that induced in COVID-19. Supported by NIH grants P01AT003961, P20GM103641, R01ES003961, R01AI129788, R01AI123947, R01AI160896 to MN and PSN and K99GM147910 to KW.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.
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Background: Infection with SARS-CoV-2 triggers reprogramming through global transcriptomic changes that drive the development of Coronavirus disease 2019 (COVID-19). Although the expression and functions of proteincoding transcripts have been widely studied in SARS-CoV-2 infection, most of the transcriptome consists of non-protein-coding RNAs (ncRNAs). Long noncoding RNAs (lncRNAs), which constitute a large proportion of the transcriptome, regulate immune responses and play prominent roles in health and disease. However, the impact of lncRNAs on SARS-CoV-2 infection is poorly understood. Our study will provide fundamental insights into the role of lncRNAs in SARS-CoV-2 infection. Method(s): We hypothesized that SARS-CoV-2-induced lncRNAs are critical regulators of viral replication and immune response. To test our hypothesis, we identified lncRNAs with significant differential expression in SARS-CoV-2 infected vs. uninfected cells across two cell types (A549-hACE2 and Calu) from published transcriptome data. We silenced the expression of the top lncRNA Bre- AS1 (BA) a human lung epithelial cell model (A549 cells stably expressing hACE2 and hTMPRSS2, A549AT) using lncRNA-specific ASO (lncsi) or negative control (NC) and compared viral replication in lncsi vs. NC cells. BA-silencing (BA-si) increased SARS-CoV-2 replication. and inhibited the expression of antiviral interferon-stimulated genes (ISG). (Tyr 705) pSTAT3 forms suppressor molecular complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) that inhibit ISG transcription. Using molecular methods such as gene-silencing, immunoprecipitation, western blot, and measuring promoter activity, we further show that Bre-AS1 inhibits the phosphorylation of STAT3 and enhances ISG transcription. Result(s): Our data show that cellular lncRNA, Bre-AS1 enhances antiviral interferon-stimulated genes (ISG) expression and inhibits replication of SARSCoV- 2. Our data show that Bre-AS1 inhibits the (Tyr705) phosphorylation of STAT3 that forms ISG repressor complexes (pSTAT3-pSTAT1 or pSTAT3-PLSCR2) and thus enhances ISG transcription. Conclusion(s): Cellular lncRNA Bre-AS1 enhances expression of antiviral interferon-stimulated genes and inhibits the replication of SARS-CoV-2. Our data show that cellular lncRNAs could play significant roles in immune response and viral propagation. Thus, unraveling the mechanisms of lncRNA-mediated regulation of virus replication and immune response may lead to identifying new, highly selective therapeutic targets Bre-AS1 inhibits STAT3 phosphorylation and enhances ISG transcription.
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Obesity is associated with several diseases, including mental health. Adipose tissue is distributed around the internal organs, acting in the regulation of metabolism by storing and releasing fatty acids and adipokine in the tissues. Excessive nutritional intake results in hypertrophy and proliferation of adipocytes, leading to local hypoxia in adipose tissue and changes in these adipokine releases. This leads to the recruitment of immune cells to adipose tissue and the release of pro-inflammatory cytokines. The presence of high levels of free fatty acids and inflammatory molecules interfere with intracellular insulin signaling, which can generate a neuroinflammatory process. In this review, we provide an up-to-date discussion of how excessive obesity can lead to possible cognitive dysfunction. We also address the idea that obesity-associated systemic inflammation leads to neuroinflammation in the brain, particularly the hypothalamus and hippocampus, and that this is partially responsible for these negative cognitive outcomes. In addition, we discuss some clinical models and animal studies for obesity and clarify the mechanism of action of anti-obesity drugs in the central nervous system.Copyright © 2023 Wolters Kluwer Medknow Publications. All rights reserved.
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The proceedings contain 23 papers. The topics discussed include: Mg and skeletal system: a link to osteoporosis and osteoarthritis;a putative impact of IL-6 on the expression of magnesiotropic genes through the activation of the JAK/STAT3 pathway;magnesium in pain therapy - historical notes and current aspects;Alzheimer's-associated variant rs708727 might be connected to dementia in Parkinson's disease;effect of magnesium citrate supplementation on the brain tissue of patients with Miyoshi dysferlinopathy measured by 31P magnetic resonance spectroscopy;clinical status of magnesium implants;Ionized magnesium: update 2022;magnesium in the treatment of selected types of muscular dystrophy;magnesium speciation analysis in blood serum;epigenetically-induced modulation of the HPA axis might improve resilience to chronic stress;magnesium status in patients with fibromyalgia syndrome;and post-covid-syndrome and transient microvascular pathology in pulse-wave-analysis - association with Mg/Ca ratio and magnesium therapy-options.
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The common reported adverse impacts of COVID-19 vaccination include the injection site's local reaction followed by various non-specific flu-like symptoms. Nevertheless, uncommon cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) following viral vector vaccines (ChAdOx1 nCoV-19 vaccine, Ad26.COV2 vaccine) have been reported. This literature review was performed using PubMed and Google Scholar databases using appropriate keywords and their combinations: SARS-CoV-2, adenovirus, spike protein, thrombosis, thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia (VITT), NF-kappaB, adenoviral vector, platelet factor 4 (PF4), COVID-19 Vaccine, AstraZeneca COVID vaccine, ChAdOx1 nCoV-19 COVID vaccine, AZD1222 COVID vaccine, coagulopathy. The s and titles of each article were assessed by authors for screening and inclusion English reports about post-vaccine CVST and VITT in humans were also collected. Some SARS-CoV-2 vaccines based on viral vector, mRNA, or inactivated SARS-CoV-2 virus have been accepted and are being pragmatic global. Nevertheless, the recent augmented statistics of normally very infrequent types of thrombosis associated with thrombocytopenia have been stated, predominantly in the context of the adenoviral vector vaccine ChAdOx1 nCoV-19 from Astra Zeneca. The numerical prevalence of these side effects seems to associate with this particular vaccine type, i.e., adenoviral vector-based vaccines, but the meticulous molecular mechanisms are still not clear. The present review summarizes the latest data and hypotheses for molecular and cellular mechanisms into one integrated hypothesis demonstrating that coagulopathies, including thromboses, thrombocytopenia, and other associated side effects, are correlated to an interaction of the two components in the COVID-19 vaccine.Copyright © 2022, Iranian Pediatric Hematology and Oncology Society. All rights reserved.
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COVID-19 has been found to affect the expression profile of several mRNAs and miRNAs, leading to dysregulation of a number of signaling pathways, particularly those related to inflammatory responses. In the current study, a systematic biology procedure was used for the analysis of high-throughput expression data from blood specimens of COVID-19 and healthy individuals. Differentially expressed miRNAs in blood specimens of COVID-19 vs. healthy specimens were then identified to construct and analyze miRNA-mRNA networks and predict key miRNAs and genes in inflammatory pathways. Our results showed that 171 miRNAs were expressed as outliers in box plot and located in the critical areas according to our statistical analysis. Among them, 8 miRNAs, namely miR-1275, miR-4429, miR-4489, miR-6721-5p, miR-5010-5p, miR-7110-5p, miR-6804-5p and miR-6881-3p were found to affect expression of key genes in NF-KB, JAK/STAT and MAPK signaling pathways implicated in COVID-19 pathogenesis. In addition, our results predicted that 25 genes involved in above-mentioned inflammatory pathways were targeted not only by these 8 miRNAs but also by other obtained miRNAs (163 miRNAs). The results of the current in silico study represent candidate targets for further studies in COVID-19.Copyright © 2023 Elsevier B.V.
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Background: SARS-CoV-2 has led to a sharp increase in the number of hospitalizations and deaths from pneumonia and multiorgan disease worldwide;therefore, SARS-CoV-2 has become a global health problem. Supportive therapies remain the mainstay treatments against COVID-19, such as oxygen inhalation, antiviral drugs, and antibiotics. Traditional Chinese medicine (TCM) has been shown clinically to relieve the symptoms of COVID-19 infection, and TCMs can affect the pathogenesis of SARS-CoV-2 infection in vitro. Jing Si Herbal Drink (JSHD), an eight herb formula jointly developed by Tzu Chi University and Tzu Chi Hospital, has shown potential as an adjuvant treatment for COVID-19 infection. A randomized controlled trial (RCT) of JSHD as an adjuvant treatment in patients with COVID-19 infection is underway Objectives: This article aims to explore the efficacy of the herbs in JSHD against COVID-19 infection from a mechanistic standpoint and provide a reference for the rational utilization of JSHD in the treatment of COVID-19. Method(s): We compiled evidence of the herbs in JSHD to treat COVID-19 in vivo and in vitro. Result(s): We described the efficacy and mechanism of action of the active ingredients in JSHD to treat COVID-19 based on experimental evidence. JSHD includes 5 antiviral herbs, 7 antioxidant herbs, and 7 anti-inflammatory herbs. In addition, 2 herbs inhibit the overactive immune system, 1 herb reduces cell apoptosis, and 1 herb possesses antithrombotic ability. Conclusion(s): Although experimental data have confirmed that the ingredients in JSHD are effective against COVID-19, more rigorously designed studies are required to confirm the efficacy and safety of JSHD as a COVID-19 treatment.Copyright © 2021
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The SARS-CoV-2 is the betacoronavirus responsible for the coronavirus disease 2019 (COVID-19) pandemic. Severe COVID-19 affects approximately 10-15% of patients and results in prolonged morbidity and mortality. Little is known about the immunophenotypic changes of the lung parenchyma driven by the viral infection in patients who die of severe COVID-19. Ultrasound-guided lung biopsies (LB) were collected (IRB approval#1561/21) within few hours from death in 15 severe COVID-19 patients between November 2020 and January 2021, in two patients who underwent lung transplantation after COVID-19 and in one patient who had surgery for bacterial superinfection during COVID-19 disease. All samples underwent histologic and immunohistochemistry evaluation and molecular profiling using the nCounter Host Response and Coronavirus Panel plus. As controls, lungs from end-stage usual interstitial pneumonia (UIP;n=9) and from lobectomy for lung cancer (Norm;n=5) were used. Eleven lungs (61%) were positive for SARS-CoV-2 RNA. Signs of diffuse alveolar damage (DAD) were observed in 6 patients (30%). COVID-19 lungs showed a marked macrophage infiltration with M2 polarization compared with controls. Globally, COVID-19 lungs showed distinct molecular profiles from UIP or Norm lungs. Specifically, a marked upregulation of interferon-genes that was directly correlated with SARS-CoV-2 genes was seen in COVID-19 lungs. COVID-19-specific genes signatures (Log2FC >1.5;adj p<0.05) obtained using VENN diagram showed impairment of the STAT3-pathway accompanied by the upregulation of the NFkB signaling. Results herein provide new insights into lung alterations induced by severe COVID-19 and suggest novel potential targets for therapeutic intervention.
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Introduction/Background: Eosinophilic granulomatosis with polyangiitis (EGPA), previously known as Churg-Strauss Syndrome, is a rare, small to medium vessel ANCA associated vasculitis. Hallmarks of EGPA include asthma, chronic rhinosinusitis, and peripheral neuropathy. EGPA is characterized by a prodrome of asthma and allergic rhinitis, followed by peripheral blood hyper-eosinophilia and accumulation of extravascular eosinophils, and finally systemic vasculitis. Extrapulmonary involvement is common, sometimes with fatal outcomes. The onset of EPGA is typically between 25-50 years;however, EGPA also occurs during childhood and has a significant morbidity and mortality. Description/Method: Our patient presented to the emergency department with a 2-week history of lethargy, wheeze and left sided neck swelling. After testing COVID-19 positive eight months prior to this, she developed wheezy episodes and was subsequently diagnosed with asthma which was managed with bronchodilators as required. She was reviewed by an allergist who confirmed a dust mite allergy and prescribed Montelukast. She remained well during the summer months however during winter she had 3 distinctive episodes of wheeze and cough which were managed by antibiotics and prednisolone. In the emergency department, an echocardiogram was performed which showed a cardiac tamponade. She was transferred to CICU where she had a pericardial drain inserted. The fluid was abundant with inflammatory cells. Multiple investigations were performed as follows: Hb: 135g/L, wbc: 20.30 x 10 9/L, Eosinophils: 12.77 x 10 9/L, CRP: 51 mg/L, ESR: 75 mm/hr, LDH: 1188 IU/L, IgE: 8000 UI/ml, ANA, ANCA: negative. CT chest showed mediastinal lymphadenopathy and patchy bilateral infiltrate and cardiac MRI showed myopericarditis and LV fibrosis. BMA showed no malignant cells and sinusitis was confirmed by CT. On examination, she was underweight. Her nasal mucosa looked inflamed. Otherwise systemic examination was unremarkable. In the context of poor ejection fraction (20%) with LV fibrosis, urgent MDT was arranged and concluded that our working diagnosis was EGPA. The decision was made to start IV methylprednisolone 10mg/kg/day for 3 days and Ivermectin. That night our patient had a VF arrest which required a single shock conversion 4J/kg. There was 7-minute downtime. Treatment was escalated to include cyclophosphamide, rituximab and plasmapheresis. The patient made a remarkable recovery, extubated and transferred to a normal ward. Her eosinophils count and inflammatory markers improved dramatically following treatment. However, she developed severe neuropathic left leg pain and NCS confirmed peripheral neuropathy Discussion/Results: EGPA is a very rare disease and diagnosis can be challenging especially with the absence of histopathology diagnosis. Early empirical treatment especially in a very ill child in intensive care unit can save lives and divert the progress of the disease. This patient has fulfilled the American College of Rheumatology criteria to diagnose EGPA including asthma, eosinophil count > 10% of upper normal, peripheral neuropathy, pulmonary infiltrates on CT thorax and paranasal sinuses abnormalities. Cardiac biopsy of the fibrotic mass may be a useful tool for diagnosis;however, this invasive procedure may expose this patient with high risk of fatal arrhythmias. Since other causes of eosinophilia were ruled out including parasitic infections, lymphoproliferative disorders, and rare primary immunodeficiency syndromes (hyper-IgE syndrome due to STAT3 or DOCK8 deficiency and Omenn syndrome) and the patient responded well to treatment, the diagnosis of EGPA was supported. Key learning points/Conclusion: Asthma not responding to bronchodilator could be another diagnosis Eosinophilia should be interpreted with caution. Defer the need for histopathology diagnosis in critically ill children Cardiac involvement is a life-threatening marker Early diagnosis prevents life threatening complications.
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Objective To explore the application pattern and mechanism of medicine and food homologous traditional Chinese medicine (TCM) against modern viral diseases. Methods The method of literature mining was applied based on the characteristics of modern viral diseases, combining with ancient books and modern prescriptions for the prevention and treatment of viral diseases to build a relevant prescription database. Then SPSS and R language were used to analyze the high-frequency medicine and food homologous TCM and high confidence medicine and food homologous prescriptions in these prescriptions, and cluster analysis was carried out. The antiviral characteristic active ingredients of high-frequency medicinal and food homologous TCN were identified and analyzed, and the action mechanism of active ingredients against modern viral diseases was evaluate by network pharmacology. Results In the prevention and treatment of modern viral diseases, Gancao (Glycyrrhizae Radix et Rhizoma)-Chenpi (Citri Reticulatae Pericarpium)-Fuling (Poria) had the highest confidence, Glycyrrhizae Radix et Rhizoma-Jiegeng (Platycodonis Radix) had the highest support. At the same time, the prescriptions were clustered and analyzed to obtain Jinyinhua (Lonicerae Japonicae Flos)-Huangqi (Astragali Radix)-Huoxiang (Agastache rugosa), Glycyrrhizae Radix et Rhizoma-Xingren (Armeniacae Semen Amarum)-Poria-Platycodonis Radix-Citri Reticulatae Pericarpium, Ganjiang (Zingiberis Rhizoma)-Renshen (Ginseng Radix et Rhizoma), Zisu (Perilla frutescens)-Gegen (Puerariae Lobatae Radix), Lugen (Phragmitis Rhizoma)-Sangye (Mori Folium), Shengjiang (Zingiberis Rhizoma Recens)-Dazao (Jujubae Fructus) clustering new prescription. The core action targets of EGFR, CASP3, VEGFA, STAT3, MMP9, HSP90AA1, mTOR, PTGS2, MMP2, TLR4, MAPK14, etc were identified. The action mechanism involved human cytomegalovirus infection, coronavirus disease-coronavirus disease 2019 (COVID-19), etc. The core action pathway were phosphatidylinositol-3/kinase protein kinase B (PI3K/Akt) signal pathway, mitogen activated protein kinase (MAPK) signal pathway, interleukin-17 (IL-17) signal pathway, Janus kinase/signal transducer and activator of transcription (JAK/STAT) signal pathway, etc. Conclusion Through data mining, six new prescriptions for preventing and controlling modern viral diseases were obtained, and the mechanism of action was preliminarily discussed, which provided some reference for the research and development of medicine and food homologous TCM prescriptions for the prevention and treatment of viral epidemics and related health products.
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Background: Mantle cell lymphoma (MCL) is a B-cell tumor which often relapses. BCR inhibitors (Ibrutinib, Acalabrutinib) and antiapoptotic BCL2-family members blockers BH3-mimetics (Venetoclax, ABT-199) are effective drugs to fight MCL. However, the disease remains incurable, due to therapy resistance, even to the promising Venetoclax and Ibrutinib combination. Therefore, there is a profound need to explore novel useful therapeutic targets. CK2 is a S/T kinase overexpressed in several solid and blood tumors. We demonstrated that CK2, operating through a 'non-oncogene addiction' mechanism promotes tumor cell survival, and counteracts apoptosis, by activating pro-survival signaling cascades, such as NF-κ B, STAT3 and AKT. CK2 could regulate also BCL2 family members. The CK2 chemical inhibitor CX-4945 (Silmitasertib, Sil) is already under scrutiny in clinical trials in relapsed multiple myeloma, solid tumors and COVID-19. Aims: In this work, we tested the effect of CK2 chemical inhibition or knock down on Venetoclax (Ven)-induced cytotoxicity in MCL pre-clinical models to effectively reduce MCL cell growth and clonal expansion. Methods: CK2 expression and BCR/BCL2 related signaling components were analyzed in MCL cells and control cells by Western blotting. CK2 and BCL2 inhibition was obtained with Sil and Ven, respectively and with CK2 gene silencing through the generation of anti-CK2 shRNA IPTG-inducible MCL cell clones. Survival, apoptosis, mitochondrial membrane depolarization and proliferation were investigated by FACS analysis of AnnexinV/PI and JC-10 staining. The synergic action of Ven and Sil was analyzed by the Chou-Talalay combination index (CI) method. CK2 knock down in vivo was obtained in xenograft NOD-SCID mouse models Results: CK2 inactivation (with Sil or CK2 silencing) determined a reduction in the activating phosphorylation of S529 p65/RelA and S473 and S129 AKT, important survival cascades for MCL. Sil or CK2 silencing caused BCL2 and related MCL1 protein reduction, causing cell death. Importantly, we confirmed these results also in an in vivo xenograft mouse model of CK2 knockdown in MCL. Sil +Ven combination increased MCL cell apoptosis, as judged by the augmented frequency of Annexin V positive cells and expression of cleaved PARP protein, and JC-10 mitochondrial membrane depolarization, with respect to the single treatments. Captivatingly, Sil or CK2 gene silencing led to a substantial reduction of the Ven-induced increase of MCL-1, potentially counteracting a deleterious Ven-induced drawback. Analysis of cell cycle distribution confirmed an increased frequency of apoptotic cells in the sub G1 phase in CK2-silenced cells and a modulation of the other phases of the cell cycle. Remarkably, the calculated CI less than 1 suggested a strong synergic cell-killing effect between Sil and Ven, on all the cell lines tested, including those less sensitive or resistant to Ven Summary/Conclusion: We demonstrated that the simultaneous inhibition/knock down of CK2 and BCL2 synergistically cooperates in inducing apoptosis and cell cycle arrest of MCL malignant B-lymphocytes and has the potential of reducing MCL clonal growth, also counterbalancing mechanism of resistance that may arise with Ven. Therefore, CK2 is a rational therapeutic target for the treatment of MCL to be tested in combination with Ibrutinib or Ven.
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Social isolation is associated with increased risk and mortality from many diseases, such as breast cancer. Socially isolated breast cancer survivors have a 43% higher risk of recurrence and a 64% higher risk of breast cancer-specific mortality than socially integrated survivors. Since Covid-19 has dramatically increased the incidence of social isolation, it is important to determine if social isolation affects the response to endocrine therapy and/or recurrence after the therapy is completed. Since previous studies indicate that social isolation increases circulating inflammatory cytokines, we investigated if an anti-inflammatory herbal mixture Jaeumkanghwa-tang (JGT) prevents the adverse effects of social isolation on breast cancer mortality. Estrogen receptor positive mammary tumors were initiated with 7,12-dimethylbenz[a]anthracene. When a rat developed a palpable mammary tumor, it was either socially isolated (SI) by housing it singly or a rat was allowed to remain group-housed (GH). Tamoxifen (340ppm via diet) or tamoxifen + JGT (500ppm via drinking water) started when the first mammary tumor reached a size of 11 mm in diameter. Tamoxifen administration ended when a complete response to this therapy had lasted for 9 weeks (corresponds to 5 years in women). During tamoxifen therapy, social isolation non-significantly reduced the rate of complete responses to 21%, from 31% in GH group (p>0.05). After the therapy was completed, SI significantly increased local mammary tumor recurrence (p<0.001;45% GH vs 75% SI). RNAseq analysis was performed in the mammary glands. Gene set enrichment analysis (GSEA) of transcriptome showed that the increased recurrence risk in socially isolated rats was associated with an enrichment of IL6/JAK/STAT3 signaling: this result was confirmed in the tumors. In addition, oxidative phosphorylation (OXPHOS) pathway was suppressed: the suppressed genes included those involved in mitochondrial pyruvate transport and conversion of pyruvate to acetyl CoA as well as genes in the TCA cycle and mediating electron transport in mitochondrial complexes I-IV. Social isolation also increased the expression of inflammatory receptor for advanced glycation end-products (RAGE) (p≤0.05). Consumption of an anti-inflammatory JGT inhibited IL6/JAK/STAT3 signaling, upregulated OXPHOS signaling and prevented the increased risk of mammary cancer recurrence in socially isolated animals. The percentage of recurrences in the SI rats dropped from 75% without JGT to 22% with JGT (p<0.001). Breast cancer mortality among socially isolated survivors may be most effectively prevented by focusing on the period following endocrine therapy using tools that inhibit IL6/JAK/STAT3 inflammatory cytokine signaling and correct disrupted OXPHOS and mitochondrial dysfunction.
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The coronavirus disease 2019 (COVID-19) was reported in Wuhan, China, in late December 2019 and soon became the most serious global health challenge due to the high rate of human-to-human transmission. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a single-stranded RNA virus and belongs to the large Coronaviridae family [1]. The pathogenesis of the COVID-19 still remains poorly understood. Cytokine storm, a hyper-inflammatory state, is considered one of the most important causes of respiratory distress syndrome (ARDS) and death in patients with COVID-19. Clinical studies have reported that there is a strong association between the level of inflammatory cytokines and the severity of the COVID-19 [2]. The prognosis of the COVID-19 is good in most patients;however, in a small number of patients, it develops into ARDS and subsequently, death within a short time [1]. Given that there is no specific antiviral drug for the treatment of the disease, suppression of cytokine storms using FDA-approved drugs with multiple mechanisms of action may reduce the COVID-19-related mortality.
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Objective: The Chinese herbal formula Huo-Xiang-Zheng-Qi (HXZQ) is effective in preventing and treating coronavirus disease 19 (COVID-19) infection;however, its mechanism remains unclear. This study used network pharmacology and molecular docking techniques to investigate the mechanism of action of HXZQ in preventing and treating COVID-19. Methods: The Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to search for the active ingredients and targets of the 10 traditional Chinese medicines (TCMs) of HXZQ prescription (HXZQP). GeneCards, Online Mendelian Inheritance in Man (OMIM), Pharmacogenomics Knowledge Base (PharmGKB), Therapeutic Target Database (TTD), and DrugBank databases were used to screen COVID-19-related genes and intersect them with the targets of HXZQP to obtain the drug efficacy targets. Cytoscape 3.8 software was used to construct the drug-active ingredient–target interaction network of HXZQP and perform protein–protein interaction (PPI) network construction and topology analysis. R software was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Finally, AutoDock Vina was utilized for molecular docking of the active ingredients of TCM and drug target proteins. Results: A total of 151 active ingredients and 250 HXZQP targets were identified. Among these, 136 active ingredients and 67 targets of HXZQP were found to be involved in the prevention and treatment of COVID-19. The core proteins identified in the PPI network were MAPK1, MAPK3, MAPK8, MAPK14, STAT3, and PTGS2. Using GO and KEGG pathway enrichment analysis, HXZQP was found to primarily participate in biological processes such as defense response to a virus, cellular response to biotic stimulus, response to lipopolysaccharide, PI3K-Akt signaling pathway, Th17 cell differentiation, HIF-1 signaling pathway, and other signaling pathways closely related to COVID-19. Molecular docking results reflected that the active ingredients of HXZQP have a reliable affinity toward EGFR, MAPK1, MAPK3, MAPK8, and STAT3 proteins. Conclusion: Our study elucidated the main targets and pathways of HXZQP in the prevention and treatment of COVID-19. The study findings provide a basis for further investigation of the pharmacological effects of HXZQP.
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Background and purpose: Increased inflammatory cytokines, including interleukin 6 (IL-6), are associated to enhanced arrhythmogenic risk, including atrial fibrillation [1]. Moreover, direct effects of cytokines on ion channels are emerging as important mediators of arrhythmogenic remodeling [2]. In line with this, enhanced arrhythmogenesis in COVID-19 patients is hypothesized to be driven by cytokine storms, a well demonstrated condition in this setting [3]. To dissect the underlying mechanisms explaining such an association, we evaluated the proarrhythmogenic alterations of IL-6, assessing the impact on the expression of Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, of the regulatory subunits MiRP1, and on the action potential (AP) profile in HL-1 cardiomyocytes (CMs). In human left atrial samples we studied the relation occurring between the expression levels of IL-6 and of HCN channels. In human induced pluripotent stem cell (hiPSC)-derived CMs we evaluated the acute effects of IL-6 on pacemaker activity. Methods: HL-1 CMs exposed to 50 ng/ml IL-6 or vehicle were collected after 0, 0.5, 6, 12, 24 and 48 h to study intracellular signaling, ion channel expression and AP profile. The latter was assessed through a high-throughput system allowing optical detection of APs with optical stimulation. In human atrial samples obtained from patients undergoing surgery, IL-6 and HCN mRNA expression were analyzed by quantitative RT-PCR. The acute effects on pacemaker activity were evaluated in hiPSC-derived CMs exposed to increasing concentrations of IL-6. Results: In HL1 CMs IL-6 rapidly induces STAT3 phosphorylation, demonstrating the activation of IL-6 signaling cascade. IL-6 modifies HCN channel transcript and proteins at different time points, evidencing a significant downregulation of HCN4 isoform and significant upregulations of HCN1, HCN2 and MiRP1. In line with this, in human left atrial samples, expression levels of IL-6 were linearly and directly related to HCN1 channel, while they were linearly and inversely related to HCN4. Electrophysiological recordings on HL-1 CMs showed a decreasing trend of AP amplitude and of maximum diastolic potential, while AP durations tended to increase. In hiPSC-derived CMs IL-6 reduces the frequency of AP in a concentration-dependent manner. Conclusions: Our data demonstrate that in HL-1 CMs IL-6 activates a STAT3 dependent intracellular signaling that is associated to subsequent variation of HCN channel expression and a concurrent alteration of AP profile. The relation between IL-6 and HCN1,4 expression in human samples suggests a mechanistic link between IL-6 levels and ionic channel targets, including HCN channels. The reduction of AP frequency in hiPSC-derived CMs suggests a direct interaction with ion channels. We hypothesize that these modifications may lay the basis to enhance the propensity of atria to develop arrhythmias in condition of elevate IL-6 levels.
ABSTRACT
As the delta and omicron SARS-CoV-2 variants spread across the world, more tools to fight off serious infection have been developed. COVID antiviral drugs that can be taken orally at home could cut serious illness and reduce the risk of hospitalization and death. However, significant population of people consume alcohol before the infection and use of the antiviral drugs, which could potentiate side effects of the drugs on the liver. We investigated the role of alcohol in anti-Covid drug-induced stress responses in live cells. METHODS: HepG2 cells or primary mouse hepatocytes (PMH) were pre-treated with alcohol (50 mMlow dose or 100 mMhigh dose) for 6-24 hours and then treated with the newly developed oral anti-Covid drugs: nirmatrelvir, ritonavir, molnupiravir, and remdesivir at 10- 30 lg/ml for 6-24 hours. Unfolded protein response (UPR)/ER stress molecular markers (e.g. IRE1 GRP78, PERK, Xbp1 and CHOP), Golgi stress response (GSR) markers of GCP60, HSP47 and TFE3, and STAT3 were measured after the treatments. Cell death was assessed through double staining the liver cells with Syntox Green and Hoesche's Blue. RESULTS: ER stress response as indicated by IRE1, Xbp1 and CHOP was insignificant or mild in either HepG2 or PMH treated individually with alcohol at the low dose, nirmatrelvir, ritonavir, molnupiravir, or remdesivir. Alcohol or remdesivir induced moderate GSR based on mRNA increase of GCP60, HSP47 and TFE3, which was accompanied with apparent Golgi fragmentation in either HepG2 or PMH. Cell death rates in HepG2 treated with alcohol, nirmatrelvir, ritonavir, molnupiravir, or remdesivir individually were less than 5%. Pre-exposure to alcohol combined with subsequent treatment with nirmatrelvir, ritonavir molnupiravir, or remdesivir significantly increased both ER stress and GSR markers and expression of phosphorylated STAT3 (p-STAT3). Most significantly, cell death rates in HepG2 or PMH were increased by 2- to 5-fold by pre-alcohol exposure plus ritonavir, nirmatrelvir, molnupiravir, or remdesivir. The organelle stress markers, p-STAT3 and cell death were all further increased in alcoholand anti-Covid drug-treated HepG2 or primary mouse hepatocytes that were pre-infected with the lentiviruses that were pseudotyped with the SARS-CoV-2 spike protein. CONCLUSION: Our results indicate that pre-exposure to alcohol potentiates the liver cells to anti-Covid-19 drugs induced stress responses and cell death.
ABSTRACT
Background: Interaction between HIV and SARS-CoV-2 infection has not yet been fully characterized. To this purpose, an in-vitro HIV/SARS-CoV-2 coinfection assay was set up. Furthermore, the results obtained in the in-vitro model were verified in a cohort of HIV/SARS-CoV-2 coinfected young individuals. Methods: We designed an in-vitro SARS-CoV-2/HIV coinfection. We challenged PBMCs derived from 10 healthy volunteers with 1 ng/1×106 cells of HIV-1BaL and subsequently co-cultured them with a human lung epithelial cell line (CaLu3) infected with SARS-CoV-2 at 0.015 MOI. At 96 hours post HIV-1 infection, both PBMCs and CaLu3 cells were harvested for mRNA expression and proteomic analysis. Furthermore, we enrolled 85 ART-treated HIV-vertically transmitted patients (mean age 22.4 years) followed at the Unit of Pediatric Infectious Diseases, Sacco Hospital in Milan, Italy. Real-time PCR was performed to detect SARS-CoV-2 and plasma samples were tested for anti-SARS-CoV-2-specific IgG (Euroimmun Kit). The subjects who contracted SARS-CoV-2 infection (H+/S+) were compared to the HIV-positive, SARS-CoV-2 negative ones (H+/S-) and to a cohort of SARS-CoV-2 positive, HIV-negative age-matched patients (H-/S+, mean age 22.8 years). We evaluated mRNA expression of factors involved in the anti-viral immune response on PBMCs upon stimulation with SARS-CoV-2 antigens (Quantigene Plex assay) and secreted cytokines/chemokines on plasma (Multiplex Cytokine Array). Results: We observed a significant reduction of SARS-CoV-2 replication on CaLu3 cells when exposed to HIV-pre-infected PBMCs in-vitro. IL-10 expression and production were significantly higher in the coinfected condition, in both CaLu3 cells and PBMCs. The upregulation of IL-10 was associated to higher expression levels of STAT3. In the HIV-vertically transmitted cohort, 4 out of 85 subjects contracted SARS-CoV-2 infection (H+/S+). All H+/S+ patients were asymptomatic. Similarly to the data obtained in-vitro, a significant increase in both expression and production of IL-10 emerged in comparison to H+/S-and H-/S+. Conclusion: In-vitro, a dampening in SARS-CoV-2 replication, along with a higher IL-10 mRNA expression and production, have been observed in the HIV/SARS-CoV-2 coinfected condition. Presumably, IL-10 exerted its activity through the STAT3 pathway. These results were confirmed in HIV/SARS-CoV-2 coinfected subjects in which an upregulation of IL-10 was observed. Our data might be useful defining HIV/SARS-CoV-2 coinfected young individuals pathogenesis.
ABSTRACT
Introduction: Severe COVID-19 pneumonia is characterised by respiratory and multi-organ failure in the context of marked systemic inflammation. This hyperinflammatory syndrome is reflected by the elevation of several inflammatory molecules, such as C-reactive protein (CRP), ferritin, IL-6, troponin, and D-dimer. In a subset of patients, early intervention with signal inhibitors may treat the Covid-19 hyperinflammatory syndrome before the development of acute lung injury and organ failure. We present a summary of a study protocol for a randomised controlled, multi-arm trial with two novel inflammatory signal inhibitors;Ruxolitinib (RUX) and Fostamatinib (FOS) for the treatment of Covid-19 pneumonia. RUX is an oral Janus Associated Kinase (JAK1/JAK2) inhibitor approved for the treatment of splenomegaly, myelofibrosis and polycythaemia vera. Inhibition of STAT3 downregulates IL-6 and IL-23, which are important for the inflammatory effects of Th17 cells. Further, JAK2 inhibition has been shown to reduce levels of TNFa and CRP, as well as reducing viral cellular entry and assembly. FOS is an oral spleen tyrosine kinase (SYK) inhibitor approved for the treatment of chronic immune thrombocytopenia. Studies of severe acute respiratory distress syndrome (ARDS) suggest that the pathogenesis relies on a series of SYK events leading to cytokine and chemokine release. FOS acts by inhibiting SYK activity, blocking the production and release of cytokines induced via C-lectin receptors and Fc receptor activation, ameliorating the cytokine storm which precedes ARDS. Primary Objective: The primary objective of MATIS is to determine the efficacy of RUX or FOS compared to standard of care (SOC) to reduce the proportion of hospitalised patients progressing from mild or moderate to severe COVID-19 pneumonia at 14 days from baseline. Secondary objectives at 7, 14 and 28 days: - Determine the efficacy of RUX or FOS to reduce mortality - Determine the efficacy of RUX or FOS to reduce the need for invasive ventilation or ECMO - Determine the efficacy of RUX or FOS to reduce the need for non-invasive ventilation - Determine the efficacy of RUX or FOS to reduce the proportion of patients suffering significant oxygen desaturation - Determine the efficacy of RUX or FOS to reduce the need for renal replacement therapy - Determine the efficacy of RUX and FOS to reduce the incidence of venous thromboembolism COVID-19 pneumonia - Determine the efficacy of RUX and FOS to reduce the severity of COVID-19 pneumonia [graded by a modified WHO Ordinal Scale] - Determine the efficacy of RUX or FOS to reduce the level of inflammatory biomarkers - Determine the efficacy of RUX or FOS to reduce the duration of hospital admission - Evaluate the safety of RUX and FOS for COVID-19 pneumonia Study Design: This is a multi-arm, two-stage, open-label, randomised (1:1:1) controlled trial. Participants will be recruited during hospitalisation for COVID-19 in multiple centres in the UK. Eligible participants (table 1) are randomised to one of the three interventions (RUX, FOS, SOC) by a central web-based randomisation service. This uses randomisation sequences with random block sizes, stratified by age (<65 and ≥65 years) and site. The treatment duration is 14 days from baseline. Patients receiving RUX will be administered 10mg BD for Day 1-7 and 5mg BD for Day 8-14. FOS will be administered as 150mg BD day 1-7 and 100mg BD day 8-14. Participants receive follow up assessments on days 7, 14 and 28 after the first dose. Outcomes: Primary endpoints will be assessed with a pairwise comparison (FOS vs SOC and RUX vs SOC) of the proportion of participants diagnosed with severe COVID-19 pneumonia within 14 days. Severe COVID-19 pneumonia is defined by a modified WHO COVID-19 Ordinal Score 5, comprising the following indicators of disease severity: - Death - Requirement for invasive ventilation - Requirement for non-invasive ventilation including CPAP or high flow oxygen - O2 saturation < 90% on 60% inspired oxygen Samples size: In stage 1 of this multi-arm study, 171 parti ipants will be randomised (57 per arm). Following an interim analysis, if either intervention shows a signal of efficacy, stage 2 will recruit a further 95 participants per arm (Fig 1). Trial Status: Recruitment is ongoing and commenced 2nd October 2020. Currently 127 patients are recruited and stage 1 is projected to be completed by 1st September 2021. The full protocol can be accessed via the trial's website. [Formula presented] Disclosures: Milojkovic: Novartis: Honoraria, Speakers Bureau;Incyte: Honoraria, Speakers Bureau;Bristol-Myers Squibb: Honoraria, Speakers Bureau;Pfizer: Honoraria, Speakers Bureau. Cooper: Principia and Sanofi: Consultancy;Sanofi and Principia: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations expenses. OffLabel Disclosure: Fostamatanib - is a tyrosine kinase inhibitor with activity against spleen tyrosine kinase (SYK). In the context of treating COVID-19, Fostamatanib acts by inhibiting SYK activist, blocking the production and release of cytokines induced via C-lectin receptors and Fc receptor activation, ameliorating the cytokine storm which precedes ARDS. Studies of severe acute respiratory syndrome induced by coronavirus, suggest that pathogenesis relies on a series of SYK events. SYK medicates ctuokine and chemokine release, induced by the activation of C-lectin receptors and immunoglobulin Fc receptors, resulting in neutrophil and monocyte lung ingress, sequential activation of neutrophil extracellular traps and activation of lung epithelium and multiple myeloid cell. This is followed by inflammation and tissue destruction that contribute to ARDS. Ruxolitinib - A JAK1/JAK2 inhibitor. JAK and STAT molecules are proteins that trance extracellular stimulation into intracellular signalling, leading to expression of host inflammatory cytokines and a variety of immune cells. In the context of MATIS, we use low dose ruxolitinib to treat COVID-19 by targeting key signalling pathways implicated in the hyper-inflammatory response of patients with COVID-19 infection. The mechanisms of Ruxolitinib to act in COVID-19 is through inhibition of STAT3 activation, down regulating IL-6 and IL-23, signalling important for the inflammatory effects of Th17 cells. Furthermore it leads to reductions of TNFa and CRP.