MTBP enhances the activation of transcription factor ETS-1 and promotes the proliferation of hepatocellular carcinoma cells.

Increasing evidence indicates that the oncoprotein murine double minute (MDM2) binding protein (MTBP) can be considered a pro-oncogene of human malignancies; however, its function and mechanisms in hepatocellular carcinoma (HCC) are still not clear. In the present work, our results demonstrate that MTBP could function as a co-activator of transcription factor E26 transformation-specific sequence (ETS-1), which plays an important role in HCC cell proliferation and/or metastasis and promotes proliferation of HCC cells. Using luciferase and real-time polymerase chain reaction (qPCR) assays, MTBP was found to enhance the transcription factor activation of ETS-1. The results from chromatin co-immunoprecipitation showed that MTBP enhanced the recruitment of ETS-1 to its downstream gene's (mmp1's) promoter region with ETS-1 binding sites. In cellular and nude mice models, overexpression of MTBP was shown to promote the proliferation of MHCC97-L cells with low endogenous MTBP levels, whereas the knockdown of MTBP led to inhibition of the proliferation of MHCC97-H cells that possessed high endogenous levels of MTBP. The effect of MTBP on ETS-1 was confirmed in the clinical specimens; the expression of MTBP was positively correlated with the downstream genes of ETS-1, mmp3, mmp9, and uPA. Therefore, by establishing the role of MTBP as a novel co-activator of ETS-1, this work expands our knowledge of MTBP or ETS-1 and helps to provide new ideas concerning HCC-related research.

Retrospective single center study of inactivating coronavirus disease 2019 vaccine in liver transplantation recipients / 中华器官移植杂志

Objective:To explore the safety of inactivating coronavirus disease 2019(covid-19)vaccine in liver transplantation(LT)recipients.Methods:Retrospective analysis was performed for clinical data of 151 LT recipients from March 2003 to October 2019.They had stable conditions and completed the course of covid-19 vaccine.Frequencies of pain at injection site, fatigue, headache and pruritus after vaccination were recorded.The safety profiles were compared between recipients with and without local and general adverse reactions after vaccination.At the same time, recipients completing two doses of covid-19 vaccines were grouped.According to vaccine companies, they were classified into Sinovac Biotech Ltd and Beijing Biological.Based upon more than or less than 60 years, they were grouped into <60 years and ≥60 years.The safety profiles of inactivating COVID-19 vaccine were compared in subgroups.Results:Among 151 eligible LT recipients, 98 of them were in group of age <60 years and 53 in group of age >60 years.The median period between vaccination and LT was 8.44(4.37, 12.39)years and the median concentration of tacrolimus 2.5(1.8, 3.9)ng/L.Eighty-three cases completed two doses of Sinovac Biotech Ltd(Sinovac Biotech Ltd group)and 40 cases Beijing Biological(Beijing Biological group); 14 cases had combined course of Sinovac Biotech Ltd and Beijing Biological, four recipients were vaccinated with inactivated vaccine from other companies and ten recipients did not know their inactivated vaccine' companies.After immunization, 24/151(15.9%)recipients had a local and general adverse reaction.The prevalence of pain at injection site, fatigue, headache and pruritus was 9.9%( n=15), 5.2%( n=8), 1.3%( n=2)and 0.7%( n=1)respectively.No significant differences existed in age( P=0.602), gender( P=0.752), period after LT( P=0.890), trough concentration of tacrolimus( P=0.377)or versions of covid-19 vaccine( P=0.582)between 24 cases with general adverse reaction and 127 without.Local and general reactions occurred in 16/83(19.3%)in Sinovac group and 5/40(12.5%)in Beijing Biological.There was no significant inter-group difference( P=0.769). There were 98 cases(64.9%)in <60 years group, 17 cases(17.3%)had local and general reaction, 53 cases(35.1%)in ≥60 years group and 7 cases(13.2%)had a local and systemic reaction.There was no significant inter-group difference( P=0.507). Conclusions:Covid-19 vaccine is safe for long-term survival LT recipients with normal liver function.Few participants present with mild fatigue and pain at injection site.

Effect of circadian rhythm changes on the expression of RORs and the RORs agonist SR1078 on corneal epithelial wound repair / 中华实验眼科杂志

Objective:To investigate the effect of circadian rhythm changes on the expression of retinoic acid-related orphan receptors (RORs) and the RORs agonist SR1078 on corneal epithelial wound repair.Methods:A total of 228 SPF C57BL/6 female mice aged 6-8 weeks old were selected, and 180 mice were divided into the normal circadian rhythm group, full-day group, full-night group, 12-hour reversed circadian rhythm group and 3-week reversed circadian rhythm group, with 36 mice in each group.The remaining 48 mice were randomly divided into phosphate buffered saline (PBS) control group and SR1078 group by random number table method, with 24 mice in each group.According to grouping, the mice were placed in a light box where the light (light intensity of 300 lx) and dark time could be controlled.The light time of the normal circadian rhythm group, the PBS control group and the SR1078 group in the light box was from 7: 00 to 19: 00, and the dark time was from 19: 00 to 7: 00 the next day.According to the Zeitgeber Time method, the starting time of light at 7: 00 was recorded as ZT0, and the time of closing light at 19: 00 was recorded as ZT12.Real-time fluorescence quantitative polymerase chain reaction was used to detect the relative expression levels of RORα and RORγ mRNA at ZT1, ZT5, ZT9, ZT13, ZT17, ZT21 in the five groups.In the PBS control group and SR1078 group, a golf-like knife was used to establish the mouse corneal epithelial injury model, and the model eyes were administered with drugs once every 6 hours according to the grouping.The corneal epithelial defect area was measured with Adobe Photoshop CC2019 software, and the corneal epithelial defect rate was calculated and compared between the two groups.The correlation between the relative expression levels of RORα and RORγ mRNA in mice corneal epithelium of the five groups and corneal epithelial defect rate in the PBS control group and SR1078 group was analyzed.The corneal epithelium repair was observed by whole cornea spreading and immunofluorescence staining, and the number of corneal epithelial dividing cells in the PBS control group and the SR1078 group was calculated and compared.The use and care of animals complied with the ARVO statement.This study protocol was approved by the Laboratory Animal Ethics Committee of Jinan University (No.JN-A-2002-01).Results:Compared with the normal circadian rhythm group, the relative expression levels of RORα/RORγ mRNA in the full-day group, full-night group, 12-hour reversed cirdian rhythym group and 3-week reversed cirdian rhythym group showed an overall decreasing trend.There was a statistically significant difference in the corneal epithelial defect rate between the PBS control group and the SR1078 group at different time points after modeling ( Fgroup=74.01, P<0.001; Ftime=5 171.48, P<0.001). Twelve hours after modeling, the corneal epithelial defect rate in the SR1078 group was significantly lower than that in the PBS control group, and the difference was statistically significant ( P<0.05). The relative expression levels of RORα and RORγ mRNA in corneal tissue was moderately positively correlated with the corneal epithelial defect rate in mice ( r=0.614, 0.537; both at P<0.01); The regression equation of the straight line between the relative expression level of RORα mRNA and the change in corneal epithelial defect rate was Y=33.153X-43.052 ( F=20.58, P<0.001), and the linear regression equation between the relative expression level of RORγ mRNA and the change of corneal epithelial defect rate was Y=2.764X-1.364 ( F=13.11, P<0.001). There was a significant overall difference in the number of corneal epithelial dividing cells at various time points following modeling between the PBS control group and the SR1078 group ( Fgroup=160.55, P<0.001; Ftime=83.57, P<0.001). The number of dividing cells in the SR1078 group was significantly less than that in the PBS control group at 24, 30, and 36 hours following modeling, and the differences were statistically significant (all at P<0.05). Conclusions:Circadian rhythm changes reduce the expression of RORα and RORγ mRNA in the mouse cornea.SR1078 can promote the expression of RORα and RORγ mRNA in corneal epithelium to decrease the number of mouse corneal epithelial dividing cells, and inhibit the repair after corneal trauma.

Rapid acquisition of a three-dimensional image of corneal nerve distribution and its various parameters / 中华实验眼科杂志

Objective:To investigate a rapid protocol for the acquisition of a three-dimensional (3D) image of corneal nerve distribution and its various parameters.Methods:Four SPF female C57BL/6 mice were selected and four corneal samples with complete limbi were obtained using a dissecting microscope after the sacrifice of mice euthanized by ether.After conventional fixation, permeabilization, and immunostaining by an anti-β-Ⅲ tubulin fluorescent-conjugated antibody, a whole-nerve image of the whole-mount cornea was captured under a 60X oil lens using a scientific complementary metal-oxide-semiconductor detector and a high-resolution deconvolution microscope.The 3D image of the corneal nerve fiber was obtained after 3D deconvolution processing, Z-axis data projection, and automatic stitching using the self-contained image processing software of the microscope system.The corneal nerve density in different areas was analyzed using the automatic detection mode in the Filament Tracer module and the manual Autopath module of the interactive microscopic image analysis software Imaris.The use and care of animals complied with the statement of the Association for Research in Visual and Ophthalmology, and the study protocol was approved by an Experimental Animal Welfare Committee of Jinan University (No.JN-A-2002-01).Results:It was found that stromal nerve fibers in a dense network entered the Bowman membrane near the limbus, and branches of stromal nerve fibers formed subbasal nerve plexus, which stretched toward the center of the cornea to form a dense neural network-like structure and converged into a vortex-like structure at the apex of the cornea.Some subbasal nerves entered the epithelial layer vertically and some branches of nerve endings were found.Through the automatic detection mode of Imaris software, a gradual increase of the density from (2 488.88±282.84)μm/μm 2 at the limbus to (5 766.66±298.55)μm/μm 2 at the center of the cornea of the subbasal nerve branches, and a decrease of the density from (40.99±0.99)μm/μm 2 at the limbus to (34.57±1.28)μm/μm 2 at the center of the stromal nerves were found.It was also found that the stromal nerves at the limbus entered the Bowman membrane for about 151 μm and then began to branch to form subbasal nerves. Conclusions:The high-resolution deconvolution microscope system can be used to study the 3D distribution of the whole corneal nerve.Additionally, Imaris can be used for obtaining various parameters of the corneal nerves automatically and quickly.

A mucosal antibody response is induced by intra-muscular SARS-CoV-2 mRNA vaccination

Although SARS-CoV-2 infects the upper respiratory tract, we know little about the amount, type, and kinetics of antibodies (Ab) generated at this site in response to intramuscular COVID-19 vaccination, and whether these Ab protect against subsequent "breakthrough" infections. We collected longitudinal serum and saliva samples from participants receiving two doses of mRNA COVID-19 vaccines over a 6-month period and measured the relative level of anti-Spike and anti-Receptor Binding Domain (RBD) Ab. We detected anti-Spike/RBD IgG and IgA and associated secretory component in the saliva of most participants receiving 1 dose of mRNA vaccine. Administration of a second dose of mRNA boosted the IgG but not the IgA response, with only 30% of participants remaining positive for IgA at this timepoint. At 6 months post-dose 2, these participants exhibited greatly diminished anti-Spike/RBD IgG and IgA levels concomitant with a reduction in neutralizing activity in the saliva, although the level of secretory component associated anti-Spike was less susceptible to decay. Examining two prospective cohorts of subjects that were monitored for infections post-vaccination, we found that participants who were subsequently infected with SARS-CoV-2 had lower levels of vaccine-induced serum anti-Spike/RBD IgA at 2-4 weeks post-dose 2 compared to participants who did not experience an infection, whereas IgG levels were comparable between groups. These data emphasize the importance of developing COVID-19 vaccines that elicit a durable IgA response. One-Sentence SummaryOur study delves into whether intra-muscular mRNA vaccination regimes confer a local IgA response in the oral cavity and whether the IgA response is associated with protection against breakthrough infection.

Immunity to SARS-CoV-2 persists 9 months post-symptoms with an altered T cell phenotype compared to influenza A virus-specific memory

SARS-CoV-2 induces T cell, B cell and antibody responses that are detected for several months in recovered individuals. Whether this response resembles a typical respiratory viral infection is a matter of debate. Here we followed T cell and antibody responses in 24 mainly non-hospitalized SARS-CoV-2 recovered subjects at two time points (median of 45- and 145-days post-symptom onset). Antibody responses were detected in 95% of subjects, with a strong correlation between plasma and salivary anti-S and anti-RBD IgG, as well as a correlation between circulating T follicular helper cells and the SARS-CoV-2-specific IgG response. Based on intracellular cytokine production or proliferation, CD4+ T cell responses to SARS-CoV-2 were detected in all subjects, decaying with a half-life of 5-6 months for S-specific IL-2-producing cells. CD4+ responses were largely of the T helper 1 phenotype, but with a lower ratio of IFN-{gamma}: IL-2 producing cells and a lower frequency of CD8+: CD4+ T cells compared to influenza A virus-(IAV)-specific memory responses within the same subjects. Analysis of secreted molecules also revealed a lower ratio of IFN-{gamma}: IL-2 and IFN-{gamma}: IL-6 and an altered cytotoxic profile for S- and N-specific compared to IAV-specific responses. These data suggest that the memory T-cell phenotype after a single infection with SARS-CoV-2 persists over time, with an altered cytokine and cytotoxic profile compared to long term memory to IAV within the same subjects. One Sentence SummaryImmunity to SARS-CoV-2 in a cohort of patients, mainly with mild COVID-19 disease, persists to 9 months with an altered T cell cytokine and cytotoxicity profile compared to influenza A virus-specific memory T cells from the same subjects.

SPEEDS: A Portable Serological Testing Platform for Rapid Electrochemical Detection of SARS-CoV-2 Antibodies

The COVID-19 pandemic has resulted in a worldwide health crisis. Rapid diagnosis, new therapeutics and effective vaccines will all be required to stop the spread of COVID-19. Quantitative evaluation of serum antibody levels against the SARS-CoV-2 virus provides a means of monitoring a patients immune response to a natural viral infection or vaccination, as well as evidence of a prior infection. In this paper, a portable and low-cost electrochemical immunosensor is developed for the rapid and accurate quantification of SARS-CoV-2 serum antibodies. The immunosensor is capable of quantifying the concentrations of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies against the SARS-CoV-2 spike protein in human serum. For IgG and IgM, it provides measurements in the range of 10.1 ng/mL - 60 {micro}g/mL and 1.64 ng/mL - 50 {micro}g/mL, respectively, and both antibodies can be assayed in 13 min. We also developed device stabilization and storage strategies to achieve stable performance of the immunosensor within 24-week storage at room temperature. We evaluated the performance of the immunosensor using COVID-19 patient serum samples collected at different time points after symptom onset. The rapid and sensitive detection of IgG and IgM provided by our immunosensor fulfills the need of rapid COVID-19 serology testing for both point-of-care diagnosis and population immunity screening.

Intranasal HD-Ad Vaccine Protects the Upper and Lower Respiratory Tracts of hACE2 Mice against SARS-CoV-2

The COVID-19 pandemic has affected more than 120 million people and resulted in over 2.8 million deaths worldwide. Several COVID-19 vaccines have been approved for emergency use in humans and are being used in many countries. However, all of the approved vaccines are administered by intramuscular injection and this may not prevent upper airway infection or viral transmission. Here, we describe intranasal immunization of a COVID-19 vaccine delivered by a novel platform, the helper-dependent adenoviral (HD-Ad) vector. Since HD-Ad vectors are devoid of adenoviral coding sequences, they have a superior safety profile and a large cloning capacity for transgenes. The vaccine (HD-Ad_RBD) codes for the receptor binding domain (RBD) of the SARS-CoV-2 spike protein and intranasal immunization induced robust mucosal and systemic immunity. Moreover, intranasal immunization of K18-hACE2 mice with HD-Ad_RBD using a prime-boost regimen, resulted in complete protection of the upper respiratory tract against SARS-CoV-2 infection. As such, intranasal immunization based on the HD-Ad vector promises to provide a powerful platform for constructing highly effective vaccines targeting SARS-CoV-2 and its emerging variants.

Mechanism of Ganshuang granule extract in alleviating N-acetyl-p-aminophenol-induced hepatocellular injury / 临床肝胆病杂志

ObjectiveTo investigate the ability of Ganshuang granule (a liver-protecting drug widely used in clinical practice) extract to reduce N-acetyl-p-aminophenol (APAP)-induced hepatotoxicity and possible mechanisms. MethodsA total of five cell culture groups were set up in this experiment, i.e., normal control group, APAP injury group, and three injury protection groups treated with different concentrations of Ganshuang granule extract. Then 20 mmol/L APAP was added to the cell culture medium and incubated for 24 hours to establish an in vitro model of drug-induced liver injury, and the injury protection groups were treated with different concentrations of Ganshuang granule extract (0.2, 1, and 5 μg/ml) in advance for 8 hours of incubation before APAP were added for 24 hours. Related markers were measured, including the markers for hepatocellular injury ［alanine aminotransferase (ALT), aspartate aminotransferase (AST), and lactate dehydrogenase (LDH)］, the markers for mitochondrial injury ［mitochondrial membrane potential, and glutamate dehydrogenase (GDH)］, and antioxidant and oxidative stress markers ［glutathione (GSH), superoxide dismutase (SOD), malondialdehyde (MDA), and reactive oxygen species (ROS)］. Related mechanism was discussed based on the experimental results. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsGanshuang granule extract alleviated APAP-induced hepatotoxicity, improved cell viability (P＜0001), and reduced the levels of AST, ALT, and LDH in supernatant (P＜0.001, P＜0.001, and P＜0.05). Ganshuang granule extract inhibited APAP-induced hepatocellular oxidative stress, and compared with the APAP group, the Ganshuang granule extract groups had significant reductions in the oxidative stress indicators ROS and MDA (both P＜0.01). Ganshuang granule extract alleviated the loss of mitochondrial membrane potential induced by APAP (P＜0.05) and reduced the content of the mitochondrial injury marker GDH in supernatant (P＜0.001) in a dose-dependent manner. Ganshuang granule extract inhibited the expression of CYP2E1/1A2 (both P＜0.05) and increased the expression of phase Ⅱ enzymes in hepatocytes. Ganshuang granule extract induced the expression of Nrf2 and its downstream genes NQO-1 and GCLC (all P＜0.05). ConclusionGanshuang granule extract can prevent APAP-induced hepatocellular injury through two ways. The first way is that Ganshuang granule extract downregulates the expression of CYP2E1/1A2 and thus reduces the production of NAPQI, a toxic product of APAP; the second way is that Ganshuang granule extract upregulates the expression of the detoxification pathway, which can activate Nrf2 to increase the expression of antioxidant enzymes (SOD and GSH) and phase Ⅱ enzymes and thus accelerate the harmless metabolism of APAP.

Protocol for the rapid quantitative analysis of neutrophils in mouse cornea by flow cytometry / 中华实验眼科杂志

Objective:To provide a standard protocol for the rapid quantitative analysis of neutrophils in inflamed corneas with flow cytometry.Methods:The corneal epithelium layer of 15 C57BL/6 mice (6-8 weeks old) was mechanically scraped off using a golf-like knife to generate a 2 mm wound region.The mouse corneas with intact limbus were cut out at 18 hours after abrasion.After mechanical shredding, the single cell suspension was obtained by collagenase I and DNase digestion.Then, the number of neutrophils in the corneal cells was sorted under the FACSCanto flow cytometer using the gate technique.Another 6 mice were taken and randomized into wounded group and normal group according to a random number table method, with 3 mice in each group.Corneal cell staining was performed using fluorescent-conjugated anti-mouse CD45, Ly6G, and CD11b antibodies.The number of neutrophils in the corneas of the two groups were enumerated and compared.The use and care of the animals complied with the Statement of the Association for Research in Vision and Ophthalmology (ARVO). The study protocol was approved by the Animal Ethics Committee of Medical College of Jinan University (No.JN-A-2002-01).Results:A standard procedure for detecting neutrophils in the cornea by flow cytometry was established.The ratio of CD45 + cells in the total corneal tissue cell population was (20.93±1.72)%.The Ly6G + and CD11b + double positive neutrophil population was sorted in the wounded corneal cell population.The ratios of Ly6G + and CD116 + cells in the CD45 + cells were (75.50±3.25)% and (93.40±4.53)%, respectively, and the ratio of the Ly6G + and CD11b + double positive neutrophils in the total number of CD45 + cells was (67.33±2.80)%.In addition, the number of neutrophils recruited to the cornea at 18 hours after corneal abrasion was (151.47±10.82)%, which was higher than (15.36±1.02)% in the normal cornea ( t=21.689, P<0.01). Conclusions:Flow cytometry can quickly and accurately quantitatively analyze the neutrophil population in the wounded cornea.It provides a rapid quantitative analysis method to further evaluate the changes of neutrophils in corneal inflammation caused by different reasons.

Pro-apoptotic molecule Noxa mediates etoposide-induced cell death in human neuroblastoma cells / 国际儿科学杂志

Objective:To study whether Noxa mediates cell death induced by etoposide in the human neuroblastoma(NB)cells.Methods:NB cells(TB3 and TB8) were treated with different concentrations of etoposide(0, 0.125, 0.25, 0.5 0.75, 1.0 mg/L), and the cell survival was detected by CCK8 assay.After treated with etoposide, NB cells were collected at different time points, then total RNAs were isolated and RT-qPCR was performed to detect the mRNA expression of Noxa.At the same time, the whole cell lysates were extracted and western blot was performed to detect the protein expression of Noxa.In order to evaluated the effect of Noxa on etoposide-induced cell survival, Noxa siRNA was transfected into NB cells, then CCK8 assay was performed.Results:After treatment with different concentrations of etoposide(0.125 mg/L、0.25 mg/L、0.5 mg/L、0.75 mg/L、1.0 mg/L ), the survival rates of TB3 cells were(73.13±8.45)%, (56.18±10.50)%, (33.90±4.17)%, (26.76±6.67)%, (13.49±0.58)%, respectively(compared with the control group, P<0.01); the survival rates of TB8 cells were(71.06±6.96)%, (37.45±0.68)%, (25.53±3.70)%, (20.28±2.75)%, (10.09±2.52)%, respectively(compared with the control group, P<0.01).The mRNA and protein expression of Noxa in NB cells were both increased in a time-dependent manner after treated with etoposide.The siRNA of Noxa could reduce the expression of Noxa in TB3 and TB8 cells after transfection.Treated with etoposide 0.5 mg/L, cell survival rates of TB3 cells tranfected with control siRNA, Noxa siRNA1, Noxa siRNA2 were(45.12±13.58)%, (72.70±21.34)%, (52.08±20.36)%, respectively; cell survival rates of TB8 were(35.52±0.38)%, (63.94±0.10)%, (50.27±1.62)%, respectively(compared with the control group, P<0.01); Treated with etoposide 1.0 mg/L, cell survival rates of TB3 cells tranfected with control siRNA, Noxa siRNA1, Noxa siRNA2 were(13.26±1.84)%, (51.08±2.41)%, (42.80±1.42)%, respectively(compared with the control group, P<0.05); cell survival rates of TB8 were(22.22±3.39)%, (58.00±11.37)%, (40.55±6.94)%, respectively(compared with the control group, P<0.05). Conclusion:Pro-apoptotic molecular Noxa mediated the Etoposide-induced cell death in NB cells(TB3 and TB8) in time and concentration-dependent manner.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations

Neutralizing antibodies (nAbs) hold promise as effective therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and its tetravalent versions can block entry with a potency that exceeds the bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, observations consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show much increased tolerance to potential virus escape mutants. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for developing potent antiviral therapies against COVID-19 and related viral threats, and our strategy can be readily applied to any antibody drug currently in development.

Systematic examination of T cell responses to SARS-CoV-2 versus influenza virus reveals distinct inflammatory profile

There is a pressing need for an in-depth understanding of immunity to SARS-CoV-2. Here we investigated T cell recall responses to fully glycosylated Spike trimer, recombinant N protein as well as to S, N, M and E peptide pools in the early convalescent phase. All subjects showed SARS-CoV-2-specific T cell responses to at least one antigen. SARS-CoV-2-specific CD4+ T cells were primarily of the central memory phenotype and exhibited a lower IFN-{gamma} to TNF- ratio compared to influenza-specific responses of the same donors, independent of disease severity. SARS-CoV-2-specific T cells were less multifunctional than influenza-specific T cells, particularly in severe cases, potentially suggesting exhaustion. High IL-10 production was noted in response to N protein, possibly contributing to immunosuppression, with potential implications for vaccine design. We observed granzyme B+/IFN-{gamma}g+ CD4+ and CD8+ proliferative responses to peptide pools in most individuals, with CD4+ responses predominating over CD8+ responses. Peripheral T follicular helper responses to S or N strongly correlated with serum neutralization assays as well as RBD-specific IgA. Overall, T cell responses to SARS-CoV-2 are robust, however, CD4+ Th1 responses predominate over CD8+ responses and are more inflammatory with a weaker Tfh response than influenza-specific CD4+ responses, potentially contributing to COVID-19 disease.

Evidence for sustained mucosal and systemic antibody responses to SARS-CoV-2 antigens in COVID-19 patients

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the mucosal immune response and its relationship to systemic antibody levels. Since SARS-CoV-2 initially replicates in the upper airway, the antibody response in the oral cavity is likely an important parameter that influences the course of infection, but how it correlates to the antibody response in serum is not known. Here, we profile by enzyme linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor binding domain (RBD) in serum (n=496) and saliva (n=90) of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Whereas anti-CoV-2 IgA and IgM antibodies rapidly decayed, IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. In a surrogate neutralization ELISA (snELISA), neutralization activity peaks by 31-45 days PSO and slowly declines, though a clear drop is detected at the last blood draw (105-115 days PSO). Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that systemic and mucosal humoral IgG antibodies are maintained in the majority of COVID-19 patients for at least 3 months PSO. Based on their correlation with each other, IgG responses in saliva may serve as a surrogate measure of systemic immunity. One Sentence SummaryIn this manuscript, we report evidence for sustained SARS-CoV-2-specific IgG and transient IgA and IgM responses both at the site of infection (mucosae) and systemically in COVID-19 patients over 3 months and suggest that saliva could be used as an alternative biofluid for monitoring IgG to SARS-CoV-2 spike and RBD antigens.

A simple protein-based SARS-CoV-2 surrogate neutralization assay

Most of the patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mount a humoral immune response to the virus within a few weeks of infection, but the duration of this response and how it correlates with clinical outcomes has not been completely characterized. Of particular importance is the identification of immune correlates of infection that would support public health decision-making on treatment approaches, vaccination strategies, and convalescent plasma therapy. While ELISA-based assays to detect and quantitate antibodies to SARS-CoV-2 in patient samples have been developed, the detection of neutralizing antibodies typically requires more demanding cell-based viral assays. Here, we present a safe and efficient protein-based assay for the detection of serum and plasma antibodies that block the interaction of the SARS-CoV-2 spike protein receptor binding domain (RBD) with its receptor, angiotensin converting-enzyme 2 (ACE2). The assay serves as a surrogate neutralization assay and is performed on the same platform and in parallel with an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against the RBD, enabling a direct comparison. The results obtained with our assay correlate with those of two viral based assays, a plaque reduction neutralization test (PRNT) that uses live SARS-CoV-2 virus, and a spike pseudotyped viral-vector-based assay.

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.

Performance verification of detecting COVID-19 specific antibody by using four chemiluminescence immunoassay systems

BackgroundThe purpose of current study is to evaluate the analytical performance of seven kits for detecting IgM/IgG antibody of corona virus (2019-nCoV) by using four chemiluminescence immunoassay systems. Methods50 patients diagnosed with 2019-nCoV infection and 130 controls without corona virus infection from the General Hospital of Chongqing were enrolled in current retrospective study. Four chemiluminescence immunoassay systems including seven IgM/IgG antibody detection Kits for 2019-nCoV (A_IgM, A_IgG, B_IgM, B_IgG, C_IgM, C_IgG, D_Ab) were employed to detecting antibody concentration. Chi-square test, receiver operating characteristic (ROC) curve and Youdens index were demonstrated to verify the cutoff value of each detection system. ResultsThe repeatability verification results of the A, B, C, and D system are all qualified. D-Ab performances best (92% sensitivity and 99.23% specificity), and B_IgM worse than other systems. Except for the system of A_IgM and C_IgG, the optimal diagnostic thresholds and cutoff value of other kits from recommendations are inconsistent with each other. B_IgM got the worst AUC and C_IgG had the best diagnostic accuracy. More importantly, B_IgG system have the highest false positive rate for testing patients with AIDS, tumor and pregnant. A_IgM system test showed highest false positive rates among elder over 90 years old. ConclusionsSystems for CoVID-2019 IgM/IgG antibody test performance difference. Serum diagnosis kit of D-Ab is the most reliable detecting system for 2019-nCoV antibody, which can be used as an alternative method for nucleic acid testing.

A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Corona Virus Disease 2019 (COVID-19)

A respiratory illness has been spreading rapidly in China, since its outbreak in Wuhan city, Hubei province in December 2019. The illness was caused by a novel coronavirus, named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical manifestations related to SARS-CoV-2 infection ranged from no symptom to fatal pneumonia. World Health Organization (WHO) named the diseases associated with SARS-CoV-2 infection as COVID-19. Real time RT-PCR is the only laboratory test available till now to confirm the infection. However, the accuracy of real time RT-PCR depends on many factors, including sampling location and of methods, quality of RNA extraction and training of operators etc.. Variations in these factors might significantly lower the sensitivity of the detection. We developed a peptide-based luminescent immunoassay to detect IgG and IgM. Cut-off value of this assay was determined by the detection of 200 healthy sera and 167 sera from patients infected with other pathogens than SARS-CoV-2. To evaluate the performance of this assay, we detected IgG and IgM in the 276 sera from confirmed patients. The positive rate of IgG and IgM were 71.4% (197/276) and 57.2% (158/276) respectively. By combining with real time RT-PCR detection, this assay might help to enhance the accuracy of diagnosis of SARS-CoV-2 infection.

Application of improved sural neuro-fasciocutaneous perforator flap harvesting and it’s application / 中华显微外科杂志

Objective:To investigate the therapeutic effect of modified sural neuro-fasciocutaneous perforator flap in reconstruction of foot and ankle soft tissue defects.Methods:Sixteen patients undergoing the modified flap for foot and ankle reconstruction were included in this study between June, 2016 and June, 2018. The 16 patients were 11 males and 5 females with an average age of 32.5 (range 21 to 51) years. Ten defects were in heel and 6 in ankle and dorsal side of foot. A "Z" -shape skin incision was performed to explore the perforator vessels. A peroneal-based perforator, a superficial vein, and the vascular axis of the sural nerve were included in the pedicle. A relaying island perforator flap was used to close the donor site without skin graft. Follow-up was carried out through outpatient service, telephone follow-up and Wechat photo transmission.Results:The patients were followed-up for 12 to 18 months. All flaps survived completely without complications. The colour, texture and apperance of the flaps were good. The area of the flaps ranged from 12 cm×5 cm to 30 cm×15 cm. The diameter of the pedicle ranged from 1 to 2 cm. No complication occurred in the donor sites. A relaying perforator island flaps were used in 10 cases for donor site closure and without a skin graft. All cases were satisfied with appearance and function at the final followed-up.Conclusion:It is possible to use the modified sural neuro-fasciocutaneous perforator flap to repair foot and ankle soft tissue defects. A relaying island perforator flap can be used as a relaying flap to cover the donor site without skin graft.

Clinical application of modified dorsal island flap of index finger for repairing the soft tissue defect of thumb tip / 中华显微外科杂志

Objective To investigate the therapeutic effect of modified dorsal metacarpal artery island flap of index finger without skin graft in repairing different types of soft tissue defect of thumb tip.Methods From August,2015 to October,2017,25 patients with soft tissue defect of the thumb tip were included in the study.Sixteen cases were males and 9 cases were females.Seventeen defects were in thumb dorsal and 8 cases were in thumb pulp.A modified dorsal island flap of index finger was used and the dorsal metacarpal superficial vein fascial flap could be harvest and combined to repair the thumb pulp defect if necessary.A relaying perforator flap pedicled on the second dorsal metacarpal artery was raised through the same incision to cover the donor site without skin graft.Followed-up was made by clinic,telephone and WeChat.Results The patients were followed-up for 6 to 18 months.All flaps survived completely without complications.The color,texture and contour of the flaps was good.Only 1 linear scar was left in the dorsum of the hand and no skin grafts.The second dorsal metacarpal artery flap was used to cover the donor site.Twenty-one cases (84％) were satisfied with the postoperative appearance of the thumb.The function was assessed as excellent in 16 fingers,good in 6 fingers and fair in 3 fingers.No complication occurred in the donor site.Conclusion It is possible to use the modified dorsal island flap of index finger to repair different types of thumb tip defects.A second dorsal metacarpal artery flap can be used as a relaying flap to cover the donor site without skin grafts.