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Meth reduces influenza A virus propagation at specific steps of the virus replication cycle. The assay to determine the effectiveness of meth in reducing influenza A virus replication with regard to the time-course of meth-exposure was performed in A549 cells as described in Materials and Methods. Cells were exposed to meth at 250 mM or left unexposed as the control (meth was not present in all incubation periods), followed by infection with influenza A/WSN/33 (H1N1) virus at an MOI of 1 PFU/cell in the absence of trypsin (single-cycle growth). Meth
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Methamphetamine (meth) is a highly addictive psychostimulant that is among the most widely abused illicit drugs, with an estimated over 35 million users in the world. Several lines of evidence suggest that chronic meth abuse is a major factor for increased risk of infections with human immunodeficiency virus and possibly other pathogens, due to its...
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... to reduce virus production from infected A549 cells. Meth was added to or excluded from the culture medium at different time periods during the course of single-cycle infection. The supernatants from infected cells were harvested at 22 h post- infection for plaque assay to determine virus yields in each meth- exposure condition. As shown in Fig. 4, when meth was added in all incubation periods (224,22 h), the virus production was significantly reduced, compared with that in the group without any meth-exposure. In the time-of-addition assay (Fig. 4B), an inhibitory effect of meth on virus production was observed when meth was present during the late post-infection (4,22 h), but ...
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... infected cells were harvested at 22 h post- infection for plaque assay to determine virus yields in each meth- exposure condition. As shown in Fig. 4, when meth was added in all incubation periods (224,22 h), the virus production was significantly reduced, compared with that in the group without any meth-exposure. In the time-of-addition assay (Fig. 4B), an inhibitory effect of meth on virus production was observed when meth was present during the late post-infection (4,22 h), but not pre-adsorption (224,0 h), adsorption (0,1 h), or early post- infection period (1,4 h). Furthermore, in the time-of-exclusion assay (Fig. 4C), a significant reduction of virus yields was found when meth ...
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... shown in Fig. 4, when meth was added in all incubation periods (224,22 h), the virus production was significantly reduced, compared with that in the group without any meth-exposure. In the time-of-addition assay (Fig. 4B), an inhibitory effect of meth on virus production was observed when meth was present during the late post-infection (4,22 h), but not pre-adsorption (224,0 h), adsorption (0,1 h), or early post- infection period (1,4 h). Furthermore, in the time-of-exclusion assay (Fig. 4C), a significant reduction of virus yields was found when meth was excluded from the medium during the pre- adsorption, adsorption, or early post-infection period, but no significant reduction was shown when meth was excluded during the late post-infection period. ...
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... in the group without any meth-exposure. In the time-of-addition assay (Fig. 4B), an inhibitory effect of meth on virus production was observed when meth was present during the late post-infection (4,22 h), but not pre-adsorption (224,0 h), adsorption (0,1 h), or early post- infection period (1,4 h). Furthermore, in the time-of-exclusion assay (Fig. 4C), a significant reduction of virus yields was found when meth was excluded from the medium during the pre- adsorption, adsorption, or early post-infection period, but no significant reduction was shown when meth was excluded during the late post-infection period. Virus production in the medium excluded before the late post-infection ...
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Background
Many flaviviruses are significant human pathogens that cause global public health threats. Developing research tools for studying and diagnosing these pathogens is a top priority. Reporter flaviviruses are useful tools for studying viral pathogenesis, diagnosing disease, and screening antiviral compounds. However, the stability of report...
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... Lysates of cultured cells and mouse brain tissues were prepared for Western blotting analysis as previously described [19,39]. For examining cell lysates, HEK293 cells were grown in 24-well plates (2×10 5 cells/well) overnight and then transfected with or without the plasmid (pAAV-αSyn; 0.5 μg/well) encoding human αSyn using the TransIT-X2 reagent (#MIR6003, Mirus). ...
Abnormal accumulation of alpha-synuclein (αSyn) in the remaining nigra dopaminergic neurons is a common neuropathological feature found in patients with Parkinson’s disease (PD). Antibody-based immunotherapy has been considered a potential approach for PD treatment. This study aims to investigate the effectiveness of active immunization against αSyn in a mouse model of PD. Adult mice were immunized with or without a synthetic peptide containing the C-terminal residues of human αSyn and activation epitopes, followed by an intranigral injection of adeno-associated virus vectors for overexpressing human αSyn. Upon the peptide injection, αSyn-specific antibodies were raised, accompanied by degeneration of dopaminergic neurons and motor deficits. Furthermore, the induction of neuroinflammation was postulated by the elevation of astroglial and microglial markers in the immunized mice. Instead of lessening αSyn toxicity, this peptide vaccine caused an increase in the pathogenic species of αSyn. Our data demonstrated the potential adverse effects of active immunization to raise antibodies against the C-terminal fragment of αSyn. This drawback highlights the need for further investigation to weigh the pros and cons of immunotherapy in PD. Applying the αSyn C-terminal peptide vaccine for PD treatment should be cautiously exercised. This study provides valuable insights into the intricate interplay among immune intervention, αSyn accumulation, and neurodegeneration.
... Western blot. Western blot analysis was performed as described previously 41,42 . The purified CD19BiTE was reconstituted in sterile PBS at a concentration of 5 ng/µl, and a portion of the reconstitution was mixed with an equal volume (25 µl) of 2 × Laemmli buffer (4% SDS, 125 mM Tris-HCl pH6.8, 10% β-mercaptoethanol, 20% glycerol, and 0.004% bromophenol blue in distilled water). ...
Blinatumomab, a bispecific T cell engager (BiTE) antibody targeting CD19 and CD3ε, can redirect T cells toward CD19-positive tumor cells and has been approved to treat relapsed/refractory B-cell acute lymphoblastic leukemia (R/R B-ALL). However, chemotherapeutic regimens can severely reduce T cells’ number and cytotoxic function, leading to an inadequate response to blinatumomab treatment in patients. In addition, it was reported that a substantial portion of R/R B-ALL patients failing blinatumomab treatment had the extramedullary disease, indicating the poor ability of blinatumomab in treating extramedullary disease. In this study, we investigated whether the adoptive transfer of ex vivo expanded γ9δ2 T cells could act as the effector of blinatumomab to enhance blinatumomab’s antitumor activity against B-cell malignancies in vivo. Repeated infusion of blinatumomab and human γ9δ2 T cells led to more prolonged survival than that of blinatumomab or human γ9δ2 T cells alone in the mice xenografted with Raji cells. Furthermore, adoptive transfer of γ9δ2 T cells reduced tumor mass outside the bone marrow, indicating the potential of γ9δ2 T cells to eradicate the extramedullary disease. Our results suggest that the addition of γ9δ2 T cells to the blinatumomab treatment regimens could be an effective approach to enhancing blinatumomab’s therapeutic efficacy. The concept of this strategy may also be applied to other antigen-specific BiTE therapies for other malignancies.
... Yemen is less than those in neighbor countries [11][12][13][14][15][16][17][18][19][20][21]. ...
... Another of the best ranked inhibitors from our computational study, methamphetamine, has actually documented good inhibitory activity against influenza A ( Chen et al., 2012). It was previously demonstrated that methamphetamine inhibits influenza A virus replication in vitro primarily via acting at the viral replication stage in which M2 plays a major role. ...
Influenza A virus (IAV) matrix protein 2 (M2), an ion channel, is crucial for virus infection, and therefore, an important anti-influenza drug target. Adamantanes, also known as M2 channel blockers, are one of the two classes of Food and Drug Administration-approved anti-influenza drugs, although their use was discontinued due to prevalent drug resistance. Fast emergence of resistance to current anti-influenza drugs have raised an urgent need for developing new anti-influenza drugs against resistant forms of circulating viruses. Here we propose a simple theoretical criterion for fast virtual screening of molecular libraries for candidate anti-influenza ion channel inhibitors both for wild type and adamantane-resistant influenza A viruses. After in silico screening of drug space using the EIIP/AQVN filter and further filtering of drugs by ligand based virtual screening and molecular docking we propose the best candidate drugs as potential dual inhibitors of wild type and adamantane-resistant influenza A viruses. Finally, guanethidine, the best ranked drug selected from ligand-based virtual screening, was experimentally tested. The experimental results show measurable anti-influenza activity of guanethidine in cell culture.
... Western blotting. Western blot analysis was performed at room temperature as reported previously 30 . ...
Methamphetamine (Meth) is one of the most frequently abused drugs worldwide. Recent studies have indicated that antibodies with high affinity for Meth reduce its pharmacological effects. The purpose of this study was to develop a technique for virus-based passive immunization against Meth effects. We generated a recombinant adeno-associated virus serotype-8 vector (AAV-MethAb) carrying the gene for a Meth-specific monoclonal antibody (MethAb). Infection of 293 cells with AAV-MethAb resulted in the expression and secretion of antibodies which bind to Meth. The viral vector was then examined in adult ICR mice. Systemic administration of AAV-MethAb resulted in long-term expression of MethAb in the serum for up to 29 weeks. Serum collected from the animals receiving AAV-MethAb retained a high specificity for (+)-Meth. Animals were challenged with Meth five weeks after viral injection. Meth levels in the brain and serum were reduced while Meth-induced locomotor activity was significantly attenuated. In conclusion, AAV-MethAb administration effectively depletes Meth from brain and serum while reducing the behavioral response to Meth, and thus is a potential therapeutic approach for Meth abuse.
With regular influenza epidemics and the emergence of drug-resistant virus strains, it is extremely crucial to develop effective and low-toxicity anti-influenza A virus drugs that act on conserved sites of novel targets. Here, we identified a novel anti-influenza compound, 1,3-dihydroxy-6-benzo [c] chromene (D715-2441), from a library of 8,026 small molecule compounds using cell-based influenza A infection assays and explored the underlying mechanisms. Our results revealed that D715-2441 possessed antiviral activities against multiple subtypes of influenza A viruses, including H1N1, H3N2, H5N1, H7N9, the clinical isolate 690 (H3 subtype) and oseltamivir-resistant strains with an NA-H274Y mutation, and suppressed the early steps in the virus replication cycle. Further mechanistic studies indicated that D715-2441 clearly inhibited viral polymerase activity and directly influenced the location of the PB2 protein. Moreover, binding affinity analyses confirmed that D715-2441 bound specifically to the PB2cap protein. Significantly, a computer-aided molecular docking and protein sequence alignment indicated that highly conserved residues in the cap-binding domain (residues 318-483) of PB2cap might be the possible binding site of D715-2441, which indicates that D715-2441 might be able to be employed as a cap-binding competitor. Moreover, the combination of D715-2441 and zanamivir possessed a remarkable synergistic antiviral effect, with a FICI value of 0.40. In conclusion, these results strongly suggest that D715-2441 has potential as a promising candidate against IAV infection. More importantly, our study offers new options for the strategic development of cap-binding inhibitors of influenza A viruses.
Dendrobine, a major component of Dendrobium nobile, increasingly draws attention to its wide applications in health care. Here we explore potential effect of dendrobine against influenza A virus and elucidate the underlying mechanism. Our results indicated that dendrobine possessed antiviral activity against influenza A viruses, including A/FM-1/1/47 (H1N1), A/Puerto Rico/8/34 H274Y (H1N1) and A/Aichi/2/68 (H3N2) with IC50 value of 3.39±0.32, 2.16±0.91, 5.32±1.68 μg/mL, respectively. Mechanism studies revealed that dendrobine inhibited early steps in the viral replication cycle. Notably, dendrobine could bind to the highly conserved region of viral nucleoprotein (NP), subsequently restraining nuclear export of viral NP and its oligomerization. In conclusion, dendrobine shows potential to be developed as a promising agent to treat influenza virus infection. More importantly, the results provide invaluable information for the fully application of Traditional Chinese Medicine named "Shihu".
Human influenza A viruses (IAVs) cause global pandemics and epidemics. These viruses evolve rapidly, making current treatment options ineffective. To identify novel modulators of IAV–host interactions, we re-analyzed our recent transcriptomics, metabolomics, proteomics, phosphoproteomics, and genomics/virtual ligand screening data. We identified 713 potential modulators targeting 199 cellular and two viral proteins. Anti-influenza activity for 48 of them has been reported previously, whereas the antiviral efficacy of the 665 remains unknown. Studying anti-influenza efficacy and immuno/neuro-modulating properties of these compounds and their combinations as well as potential viral and host resistance to them may lead to the discovery of novel modulators of IAV–host interactions, which might be more effective than the currently available anti-influenza therapeutics.
Growing evidence has indicated that opioids enhance replication of human immunodeficiency virus and hepatitis C virus in target cells. However, it is unknown whether opioids can enhance replication of other clinically important viral pathogens. In this study, the interaction of opioid agonists and human influenza A/WSN/33 (H1N1) virus was examined in human lung epithelial A549 cells. Cells were exposed to morphine, methadone or buprenorphine followed by human H1N1 viral infection. Exposure to methadone differentially enhanced viral propagation, consistent with an increase in virus adsorption, susceptibility to virus infection and viral protein synthesis. In contrast, morphine or buprenorphine did not alter H1N1 replication. Because A549 cells do not express opioid receptors, methadone-enhanced H1N1 replication in human lung cells may not be mediated through these receptors. The interaction of methadone and H1N1 virus was also examined in adult mice. Treatment with methadone significantly increased H1N1 viral replication in lungs. Our data suggest that use of methadone facilitates influenza A viral infection in lungs and might raise concerns regarding the possible consequence of an increased risk of serious influenza A virus infection in people who receive treatment in methadone maintenance programs.
Illicit stimulants, such as cocaine, amphetamine, and their derivatives (e.g., "ecstasy"), continue to exact heavy toll on health care in both developed and developing countries. The US Department of Health and Human Service reported over one million illicit drug-related emergency department visits in 2010, which was higher than any of the six previous years. Both inhaled and intravenous forms of these substances of abuse can result in a variety of acute and chronic injuries to practically every part of the respiratory tract, leading potentially to permanent morbidities as well as fatal consequences-including but not limited to nasal septum perforation, pulmonary hypertension, pneumothorax, pneumomediastinum, interstitial lung disease, alveolar hemorrhage, reactive airway disease, pulmonary edema, pulmonary granulomatosis, infections, foreign body aspiration, infections, bronchoconstriction, and thermal injuries. Stimulants are all rapidly absorbed substances that can also significantly alter the patient's systemic acid-base balance and central nervous system, thereby leading to further respiratory compromise. Mounting evidence in the past decade has demonstrated that adulterants coinhaled with these substances (e.g., levamisole) and the metabolites of these substances (e.g., cocaethylene) are associated with specific forms of systemic and respiratory complications as well. Recent studies have also demonstrated the effects of stimulants on autoimmune-mediated injuries of the respiratory tract, such as cocaine-induced midline destructive lesions. A persistent challenge to studies involving stimulant-associated respiratory toxidromes is the high prevalence of concomitant usage of various substances by drug abusers, including tobacco smoking. Now more than ever, health care providers must be familiar with the multitude of respiratory toxidromes as well as the diverse pathophysiology related to commonly abused stimulants to provide timely diagnosis and effective treatment.
