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Objective:To investigate the risk factors and establish a prediction model of primary non-response (PNR) to anti-tumor necrosis factor-α(TNF-α) monoclonal antibody in Crohn′s disease (CD) patients.Methods:From December 1, 2018 to July 31, 2022, 103 patients with CD treated with the anti-TNF-α monoclonal antibody in Renmin Hospital of Wuhan University were enrolled (modeling group), and at the same time, 109 patients with CD treated with anti-TNF-α monoclonal antibody in Zhongnan Hospital of Wuhan University were selected (validation group). The baseline clinical data of all the patients before the first treatment of anti-TNF-α monoclonal antibody were collected, which included C-reactive protein (CRP), the simplified Crohn′s disease activity index (CDAI), and modified multiplier simple endoscopic score for Crohn′s disease (MM-SES-CD), etc. Multivariate logistic regression was used to screen the independent risk factors of PNR in patients with CD treated with the anti-TNF-α monoclonal antibody, and to establish the nomograms prediction model. The area under the curve (AUC) of the receiver operating characteristic curve (ROC), the net reclassification index (NRI), integrated discrimination improvement index (IDI), and decision curve analysis (DCA) were used to evaluate the predictive efficacy and clinical application value of the prediction model. DeLong test was used for statistical analysis.Results:The results of multivariate logistic regression analysis showed that high level of CRP ( OR=1.030, 95% confidence interval (95% CI) 1.002 to 1.059), simplified CDAI ( OR=1.399, 95% CI 1.023 to 1.913), and MM-SES-CD ( OR=1.100, 95% CI 1.025 to 1.181) in baseline were independent risk factors of PNR in patients with CD treated with the anti-TNF-α monoclonal antibody ( P=0.033, 0.036 and 0.008). The results of ROC analysis showed that the AUCs of CRP, simplified CDAI, MM-SES-CD, and the prediction model in the modeling group and the validation group were 0.697(95% CI 0.573 to 0.821), 0.772(95% CI 0.666 to 0.879), 0.819(95% CI 0.725 to 0.912), 0.869 (95% CI 0.786 to 0.951) and 0.856 (95% CI 0.756 to 0.955), respectively. The AUC of the prediction model in the modeling group was greater than those of CRP and simplified CDAI, and the differences were statistically significant ( Z=3.00 and 2.75, P=0.003 and 0.006), while compared with MM-SES-CD and the validation group, the differences were not statistically significant (both P>0.05). However, compared with MM-SES-CD, the NRI and IDI of the prediction model in the modeling group were 0.205(95% CI 0.002 to 0.409, P=0.048) and 0.098(95% CI 0.022 to 0.174, P=0.011), respectively, suggesting that the predictive ability of the prediction model was better than that of MM-SES-CD. The results of DCA indicated that the prediction model had significant clinical benefits in both the modeling group and the validation group. Conclusions:A prediction model was successfully constructed based on the independent risk factors for PNR in patients with CD treated with the anti-TNF-α monoclonal antibody. After verification, the prediction model has good prediction performance and significant clinical benefits.
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Objective:To construct a recombinant herpes simplex virus 2 (HSV-2) expressing enhanced green fluorescent protein (EGFP) using clustered, regularly interspaced, short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) technology.Methods:Four strategies for inserting exogenous EGFP gene into HSV-2 genome using CRISPR/Cas9 technology were designed: (1) conventional homology-directed repair: circular two homology arm donor-mediated gene knock-in; (2) linearized single homology arm donor-mediated gene knock-in; (3) homology-independent targeted integration; (4) conventional homology-directed repair-mediated by cell lines stably expressing Cas9 and sgRNA.Results:The recombinant virus HSV-2-EGFP was successfully constructed based on the second, the third and the fourth strategies. The second strategy was the most efficient, followed by the third and the fourth strategies. The purified recombinant virus could stably express green fluorescent protein in seven passages and shared similar growth characteristics in Vero cells to the parental virus.Conclusions:Linearized single homology arm donor could increase the efficiency of gene knock-in, and cell lines stably expressing Cas9 and sgRNA could increase the efficiency of gene knock-in mediated by homology-directed repair.
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Objective:To investigate the effects of genomic location of a foreign gene in Shanghai-191 strain measles virus (MV) vector on gene expression and virus replication.Methods:The nucleotide sequence encoding S1 protein of SARS-CoV-2 was inserted at different positions in MV antigenome (the upstream of the N gene, between P and M genes, between H and L genes), and co-transfected into 293T cells with helper plasmids coding T7 RNA polymerase and N, P, and L proteins, respectively. The transfected cells were lysed and the supernatants were used to infected Vero cells to harvest recombinant viruses. S1 proteins expressed by the recombinant viruses were identified by RT-PCR, indirect immunofluorescence assay, Western blot and ELISA. Growth kinetics of the recombinant viruses were analyzed.Results:Recombinant viruses were failed to be rescued when the S1 protein-coding sequence was cloned into the upstream of N gene. Two recombinant viruses, MV-M-S1 and MV-L-S1, were successfully rescued when cloning the S1 protein-coding sequence into the intergenic region between P and M genes, or H and L genes, and could express S1 protein. MV-M-S1 expressed more S1 protein than MV-L-S1, but the titer of MV-M-S1 was lower.Conclusions:Inserting a foreign gene at different positions in the MV genome might have different effects on gene expression and virus replication. This study provided reference for the subsequent construction of MV vector.
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Herpes simplex virus (HSV), the main pathogen of human herpetic diseases, is an attractive viral vector due to a wide range of host cell types and a large capacity of foreign gene. The manipulation on herpes simplex virus genome, up to 152 kb, has been the key process and has undergone the improvement from homologous recombination to bacterial artificial chromosome (BAC) and CRISPR/Cas9. However, homologous recombination is imperfect because the efficiency of recombination is relatively low and the product is too complex to purify. Construction of recombinant HSV based on BAC is time-and labor-consuming. Currently, CRISPR/Cas9 has been applied to genetic engineering of various species by more and more researchers due to its convenience, good repeatability, low cost, and then the manipulation of viral genome is more accurate and efficient. Application of CRISPR/Cas9 in the research of HSV gene function, HSV infection therapy and viral vector were summarized in this review, in order to provide reference for corresponding research.
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Objective:To investigate the clinical characteristics of asymptomatic carriers with 2019 novel coronavirus (2019-nCoV), and to provide clinical guidance for the management of asymptomatic infection with 2019-nCoV.Methods:The clinical data of 663 patients with confirmed coronavirus disease 2019 (COVID-19) admitted to Renmin Hospital of Wuhan University from January 11 to February 6, 2020 were collected. Patients were divided into asymptomatic group (21 cases) and symptomatic group (642 cases) according to the diagnostic criteria. General conditions, clinical classification, death, chest computed tomograph (CT) and laboratory results of patients were retrospectively collected. Mann-Whitney U test, chi-square test and Fisher exact test were used for statistical analysis. Results:All 663 patients were positive for 2019-nCoV nucleic acid tests. The age of patients in the asymptomatic group were significantly younger than those in symptomatic group (35.0 (31.5, 58.0) years old vs 58.5 (45.0, 69.0) years old, U=4 234.500, P=0.002). The proportion of patients <30 years old in the two groups was significantly different (19.0%(4/21) vs 6.1%(39/642), Fisher exact test, P=0.047). There were 15 women (71.4%) in the asymptomatic group and 327 women (50.9%) in the symptomatic group, while the difference of gender distributions was not statistically significant ( χ2=3.420, P=0.064). In addition, among patients with asymptomatic infection, the proportions of mild/ordinary, severe and critical patients were 10 cases (47.6%), 10 cases (47.6%), and one case (4.8%), respectively, which were not significantly different from those in symptomatic group (244 cases (38.0%), 305 cases (47.5%) and 93 cases (14.5%), respectively, χ2=1.847, P=0.397). As of February 9, one(4.8%) mild/ordinary patient in the asymptomatic group died who had malignant tumor. Twenty-four (3.7%) patients in the symptomatic group died including two mild/ordinary and 22 critical patients. There was no significant difference in mortality between the two groups(Fisher exact test, P=0.560). CT examination was performed on 594 patients, and 591 cases (99.5%) showed unilateral or bilateral pneumonia, and three cases (0.5%) showed normal. Conclusions:Patients with asymptomatic infection with 2019-nCoV are younger than symptomatic patients, and there are more patients under 30 years old in the asymptomatic group. The absence of clinical symptoms is not significantly associated with clinical classifications and mortality in COVID-19 patients.
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Objective:To retrospectively analyze the clinical characteristics of patients with coronavirus disease 2019 (COVID-19) accompanied with diarrhea.Methods:From January 11 to February 6 in 2020, the clinical data of 663 patients diagnosed with COVID-19 admitted to Renmin Hospital of Wuhan University were collected and divided into diarrhea group and non-diarrhea group according to whether they had diarrhea or not. The differences in baseline characteristics, basic disease history, clinical manifestations, chest computed tomography (CT), laboratory findings, disease severity and mortality between the two groups were compared. Chi-square test and Fisher exact test were used for statistical analysis.Results:Among 663 COVID-19 patients, 70 (10.6%) patients accompanied with diarrhea. The proportion of fatigue and increased lactate dehydrogenase (LDH) levels of diarrhea group were higher than those of non-diarrhea group (58.6%, 41/70 vs. 28.2%, 167/593; and 64.2%, 43/67 vs. 50.4%, 277/550), and the differences were statistically significant ( χ2=26.891 and 4.566, both P<0.05). There was no statistically significant difference in the proportion of pneumonia in chest CT between diarrhea group and non-diarrhea group (100.0%, 62/62 vs. 99.4%, 529/532) ( P>0.05). There were no statistically significant differences in the proportions of mild and normal type, severe type and critical type between diarrhea group and non-diarrhea group (35.7%, 25/70 vs. 38.6%, 229/593; 50.0%, 35/70 vs. 47.2%, 280/593; and 14.3%, 10/70 vs. 14.2%, 84/593, respectively) (all P>0.05). There were no statistically significant differences in the mortality of mild and normal type, severe type and critical type between diarrhea group and non-diarrhea group (0 vs. 0.5%, 3/593; 0 vs. 0 and 1.4%, 1/70 vs. 3.5%, 21/593) (all P>0.05). Conclusions:Patients with COVID-19 accompanied with diarrhea are more likely to have fatigue and increased LDH level. Diarrhea is not significantly correlated with the disease severity of patients with COVID-19.
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Objective@#To develop a real-time quantitative PCR (qPCR) assay for detecting Sendai virus gene copy number.@*Methods@#The recombinant plasmid was constructed and a reference used to establish the real-time qPCR method with the TaqMan probe and verified for reproducibility and sensitivity.@*Results@#The linear range of the developed real-time qPCR assay was 1.024 × 103 ~5 × 1010 copies/μl, with an R2 value of more than 0. 99. The standard recovery of the tested samples was 84.56%~119.48%.@*Conclusions@#Compared with traditional hernagglutination assay for particle number detection, this method has higher sensitivity, better repeatability and high quantitative accuracy. It can be used for the detection of particle number of vaccine with sendai virus as the carrier, so as to detect the specific activity of vaccine products.
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Objective@#To explore the relationship between childhood abuse, adult attachment and borderline personality disorder (BPD) in college students, and to provide reference for promoting the physical and mental health of college students.@*Methods@#We selected undergraduate students from four colleges in Hefei, a total of 4 034 college students were surveyed by the childhood trauma questionnaire short form(CTQ-SF), the state adult attachment measure (SAAM) and the Personality Diagnostic Questionnaire(PDQ).@*Results@#BPD was found in 4.2% of subjects, 4.5% of males and 3.7% of females. The score of BPD in non-single-parent families was lower than that in single-parent families, in families with average family economic status was lower than that in families with poor and good family economic status, and in families with medium father education level was lower than that in fathers with low and high education level(Z=-2.30, 29.25, 9.63, P<0.05). Childhood abuse, avoidant attachment and anxious attachment positively predicted BPD(β=0.21, 0.10, 0.23, P<0.01), secure attachment negatively predicted BPD(β=-0.15, P<0.01). Adult attachment played a partial mediating role in the effects of childhood abuse on BPD, with the mediating effect accounting for 16.7% of the total effect.@*Conclusion@#Adult attachment plays a mediating role in the effects of childhood abuse on BPD.