Reduced neutralization of SARS-CoV-2 B.1.617 variant by inactivated and RBD-subunit vaccine

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The Spike protein that mediates coronavirus entry into host cells is a major target for COVID-19 vaccines and antibody therapeutics. However, multiple variants of SARS-CoV-2 have emerged, which may potentially compromise vaccine effectiveness. Using a pseudovirus-based assay, we evaluated SARS-CoV-2 cell entry mediated by the viral Spike B.1.617 and B.1.1.7 variants. We also compared the neutralization ability of monoclonal antibodies from convalescent sera and neutralizing antibodies (NAbs) elicited by CoronaVac (inactivated vaccine) and ZF2001 (RBD-subunit vaccine) against B.1.617 and B.1.1.7 variants. Our results showed that, compared to D614G and B.1.1.7 variants, B.1.617 shows enhanced viral entry and membrane fusion, as well as more resistant to antibody neutralization. These findings have important implications for understanding viral infectivity and for immunization policy against SARS-CoV-2 variants.

Emerging SARS-CoV-2 variants reduce neutralization sensitivity to convalescent sera and monoclonal antibodies

SARS-CoV-2 Spike-specific antibodies contribute the majority of the neutralizing activity in most convalescent human sera. Two SARS-CoV-2 variants, N501Y.V1 (also known as B.1.1.7 lineage or VOC-202012/01) and N501Y.V2 (B.1.351 lineage), reported from the United Kingdom and South Africa, contain several mutations in the receptor binding domain of Spike and are of particular concern. To address the infectivity and neutralization escape phenotypes potentially caused by these mutations, we used SARS-CoV-2 pseudovirus system to compare the viral infectivity, as well as the neutralization activities of convalescent sera and monoclonal antibodies (mAbs) against SARS-CoV-2 variants. Our results showed that N501Y Variant 1 and Variant 2 increase viral infectivity compared to the reference strain (wild-type, WT) in vitro. At 8 months after symptom onset, 17 serum samples of 20 participants (85%) retaining titers of ID50 >40 against WT pseudovirus, whereas the NAb titers of 8 samples (40%) and 18 samples (90%) decreased below the threshold against N501Y.V1 and N501Y.V2, respectively. In addition, both N501Y Variant 1 and Variant 2 reduced neutralization sensitivity to most (6/8) mAbs tested, while N501Y.V2 even abrogated neutralizing activity of two mAbs. Taken together the results suggest that N501Y.V1 and N501Y.V2 reduce neutralization sensitivity to some convalescent sera and mAbs.

Evodiamine induces apoptosis of leukemia cell line K562 VIA modulation of TRIB2/AKT pathway / 中国药理学通报

Aim To investigate the effects of Evodiamine (EVO) on proliferation and apoptosis of human leukemia cell line K562 and its potential mechanisms. Methods K562 cells were treated with EVO at different concentrations (0, 1, 2, 4, 8, 16, 32, 64 jxmol • L

Changes of Humoral Immunity Response in SARS-CoV-2 Convalescent Patients over 8 months

Many countries around the world have all seen a sharp rise in COVID-19 cases as the second wave since the beginning of October 2020. Decline of antibodies response to severe acute respiratory syndrome coronavirus (SARS-CoV-2) that was reported exclusively in the early month increases the risk of reinfection for convalescent individuals. There is a current need to follow the maintenance of special antibodies against SARS-CoV-2. Here, we reported changes of antibodies against SARS-CoV-2 in convalescent patients over 8 months. Antibodies of all 20 participants targeting SARS-CoV-2 spike receptor binding-domain (RBD) had decreased from a mean OD450 value 1.78 to 0.38 over 8 months. The neutralizing antibody (NAb) titers decreased from the mean ID50 value 836 to 170. The NAb titers were significantly correlated with IgG level during 8 months (P<0.001). Furthermore, while RBD-specific IgG existence of 25% (5/20) convalescent plasma was undetectable, the NAb titers of 15% (3/20) convalescent plasma decreased below the threshold. In addition, compared to wild-type SARS-CoV-2 (S-D614), lower titers of neutralizing antibodies against its G614 variant were shown at 8 months after symptom onset. This study has important implications when considering antibody protection against SARS-CoV-2 reinfection.

Cytokine biomarkers of COVID-19

We used a new strategy to screen cytokines associated with SARS-CoV-2 infection. Cytokines that can classify populations in different states of SARS-CoV-2 infection were first screened in cross-sectional serum samples from 184 subjects by 2 statistical analyses. The resultant cytokines were then analyzed for their interrelationships and fluctuating features in sequential samples from 38 COVID-19 patients. Three cytokines, M-CSF, IL-8 and SCF, which were clustered into 3 different correlation groups and had relatively small fluctuations during SARS-CoV-2 infection, were selected for the construction of a multiclass classification model. This model discriminated healthy individuals and asymptomatic and nonsevere patients with accuracy of 77.4% but was not successful in classifying severe patients. Further searching led to a single cytokine, hepatocyte growth factor (HGF), which classified severe from nonsevere COVID-19 patients with a sensitivity of 84.6% and a specificity of 97.9% under a cutoff value of 1128 pg/ml. The level of this cytokine did not increase in nonsevere patients but was significantly elevated in severe patients. Considering its potent antiinflammatory function, we suggest that HGF might be a new candidate therapy for critical COVID-19. In addition, our new strategy provides not only a rational and effective way to focus on certain cytokine biomarkers for infectious diseases but also a new opportunity to probe the modulation of cytokines in the immune response.

Antibody responses to SARS-CoV-2 in COVID-19 patients: the perspective application of serological tests in clinical practice

BackgroundWe aim to investigate the profile of acute antibody response in COVID-19 patients, and provide proposals for the usage of antibody test in clinical practice. MethodsA multi-center cross-section study (285 patients) and a single-center follow-up study (63 patients) were performed to investigate the feature of acute antibody response to SARS-CoV-2. A cohort of 52 COVID-19 suspects and 64 close contacts were enrolled to evaluate the potentiality of the antibody test. ResultsThe positive rate for IgG reached 100% around 20 days after symptoms onset. The median day of seroconversion for both lgG and IgM was 13 days after symptoms onset. Seroconversion of IgM occurred at the same time, or earlier, or later than that of IgG. IgG levels in 100% patients (19/19) entered a platform within 6 days after seroconversion. The criteria of IgG seroconversion and > 4-fold increase in the IgG titers in sequential samples together diagnosed 82.9% (34/41) of the patients. Antibody test aided to confirm 4 patients with COVID-19 from 52 suspects who failed to be confirmed by RT-PCR and 7 patients from 148 close contacts with negative RT-PCR. ConclusionIgM and IgG should be detected simultaneously at the early phase of infection. The serological diagnosis criterion of seroconversion or the >; 4-fold increase in the IgG titer is suitable for a majority of COVID-19 patients. Serologic test is helpful for the diagnosis of SARS-CoV-2 infection in suspects and close contacts.

Analysis of the impact of extracellular acidity on the expression and activity of P-glycoprotein and on the P-glycoprotein-mediated cytotoxicity of daunorubicin in cancer cell by microfluidic chip technology / 中国医学科学院学报

<p><b>OBJECTIVE</b>To explore the impact of extracellular acidic environment on the expression and activity of P-glycoprotein (P-gp) and on the P-gp-mediated cytotoxicity of daunomycin in cancer cells by using microfluidic chip technology.</p><p><b>METHODS</b>The A549 cells cultured on a microfluidic chip were divided into experiment group and control group. The experiment group was exposed to an acidic cell culture medium (pH 6.6), while the control group was treated with a neutral cell culture medium (pH 7.4). The expression of P-gp was detected by cell immunofluorescense analysis and the activity of P-gp was evaluated by Rhodamine 123 efflux experiment. Meanwhile, the cytotoxicity of daunomycin was analyzed by cell live/dead fluorescence staining method.</p><p><b>RESULTS</b>Microfluidic chip designed in this study could provide a suitable microenvironment for the growth of A549 cells and the A549 cells reached the confluence of 90% after inoculation for 72 h. Treatment of the acidic cell culture media on A549 cells did not make a significant difference on the expression level of P-gp. However, the activity of P-gp was significantly enhancement and peaked at 6 h after treatment with acidic cell culture media. Meanwhile, the cytotoxicity of daunomycin reduced significantly after treatment with acidic cell culture medium for 6 h,and a reversal effect was obtained when synergy with verapamil.</p><p><b>CONCLUSIONS</b>Microfluidic chip technology can shorten the analysis time and reduce the reagent consumption. It can be used as a new technology platform for understanding the mechanisms of multi-drug resistance and for screening highly efficient multi-drug resistance reversal agents.</p>

Neutrophil elastase inhibitor on proliferation and apoptosis of U937 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study and compare the effect of neutrophil elastase inhibitors (GW311616A and sivelestat) on the proliferation and apoptosis of U937 cells.</p><p><b>METHODS</b>Inhibitory effects of GW311616A and sivelestat on the proliferation of U937 cells were assayed by MTT assay. The morphologic changes of U937 cells were detected by transmission electron microscope, and apoptosis was observed by AnnexinV-FITC/PI staining. The changes of cell cycle and apoptosis were detected by flow cytometry. The expression of NE in U937 cells was observed by indirect immunofluorescence, the variations of content and activity of NE in U937 cells were measured through ELISA assay and colorimetric method.</p><p><b>RESULTS</b>MTT showed that both NE inhibitors could inhibit the proliferation of U937 cells in a dose dependent manner. The IC50 of GW311616A and sivelestat were 150 and 214 μmol/L respectively. The inhibition effect of GW311616A was significantly higher than of sivelestat (P<0.01). Typical apoptosis morphological changes of U937 cells was observed through electron microscope. AnnexinV-FITC/PI staining showed that U937 cells could be induced to undergo apoptosis by the two inhibitors, the apoptosis ratio of 150μmol/L GW311616A group (13.60%) was significantly higher than that of 150μmol/L sivelestat group (3.69%)(P<0.01). The result of flow cytometry indicated that the apoptosis ratio of 150 μmol/L GW311616A group was 14.61%, U937 cell cycle was mainly blocked in G2/M phase; meanwhile 150 μmol/L sivelestat group as 4.25% with cell cycle in S phase. The fluorescence intensity of GW311616A group obviously decreased than of sivelestat group. And the two inhibitors could reduce the content and activity of NE in U937 cells, but the effect of GW311616A was significantly higher than of sivelestat (P<0.01).</p><p><b>CONCLUSION</b>GW311616A and sivelestat could inhibit the proliferation and cause apoptosis of U937 cells. Furthermore, GW311616A was more effective and harmful to cells than sivelestat.</p>

Anti-tumor activity of emodin against human chronic myelocytic leukemia K562 cell lines in vitro and in vivo.

Emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), a natural anthraquinone derivative isolated from Rheum palmatum L, has been reported to exhibit anti-cancer effect on several human cancers such as liver cancers and lung cancers. However, the molecular mechanisms of emodin-mediated tumor regression have not been fully defined. Our preliminary study showed that emodin had highly cytotoxic effect on human chronic myeloid leukemia K562 cell lines. This study was performed to investigate the anti-tumor effect of emodin in human K562 cell line in vitro and in vivo. The MTT data showed the inhibition on growth of K562 cells following emodin treatment. Flow cytometry showed that the cell cycle of K562 cells was arrested in G(0)/G(1) phase. Through Western blot analysis, we found that the apoptosis-related protein Bcl-2 was decreased in a dose-dependent manner and the Bax was increased after emodin treatment. Moreover, activations of caspase-3, -8 and -9 were demonstrated in vitro and in vivo. The increased Bax concurrent with the decreased of Bcl-2 indicated that emodin treatment might result in apoptosis of K562 cells. The cell apoptosis was also directly demonstrated by Annexin V-FITC, and DNA fragmentation assay. Additionally, the tumoricidal effect of emodin was measured using a xenograft nude mice model. We found that, after inoculated with the K562 cells, the nude mice treated with emodin showed a significant decrease of tumor volume and tumor weight in comparison to the control. Emodin could cause the regression of tumor. Both in vitro and in vivo studies suggest that emodin can be developed as a promising anti-chronic myeloid leukemia drug.

Screening and identification of proteins interacting with RAR alpha-V via yeast two-hybrid system / 中华血液学杂志

<p><b>OBJECTIVE</b>To screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.</p><p><b>METHODS</b>The bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.</p><p><b>RESULTS</b>The bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro.</p><p><b>CONCLUSIONS</b>There are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.</p>

Protective effects of meloxicam on aluminum overload-induced cerebral damage in mice.

The putative protective effects of meloxicam on the oxidative damage induced by aluminum overload in mice brain were investigated. The cerebral damage model in mice was established via intracerebroventricular (i.c.v.) microinjection of aluminum (5.0 microg in 2.0 microl), once a day for 5 days. Meloxicam, a selective inhibitor of cyclooxygenase-2 (COX-2), was intragastrically (i.g) administered 30 min before each aluminum administration, and continuously given for another 10 days after the last aluminum administration. Behavioral changes including locomotor activity, passive avoidance, and spatial learning and memory ability were examined two weeks after the last administration of meloxicam. To determine the brain damage, we also measured pathological alterations in the cerebral tissue, malondialdehyde contents and expressions of Choline acetyltransferase (ChAT), amyloid precursor protein (APP) and amyloid beta. Furthermore, COX-2 proteins and COX-2 mRNA were examined to investigate the mechanism for underlying the effect of meloxicam. The impairment of learning and memory function was caused by aluminum overload. Consistent with the behavioral changes, neuronal death in the hippocampi, increased content of malondialdehyde, expressions of APP, amyloid beta and COX-2 proteins, as well as COX-2 mRNA, and decreased expression of ChAT protein were detected in the aluminum-overload mice. Meloxicam significantly protected mice from the brain damage, and behavioral and biochemical changes above caused by aluminum overload. These experimental results indicate that there is a close relationship between over-expression of COX-2 and neuron damage induced by aluminum overload. It also suggests that selective inhibitors of COX-2 have potential values in clinical treatment for some other neuron damage-related diseases.