Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

Objective To observe the effects ofJingui Shenqi Pills on the expressions of TGF-β1, CYP19, sex hormone level and sperm quality;To discuss its mechanism for the treatment of male sterility.Methods Forty adult male SD rats were randomly divided into normal group, model group, retardant group andJingui Shenqi Pills group, 10 rats in each group. Except for the normal group, adenine was used to induce healthy rats to kidney yang deficiency and sterility model rats. Normal group was given normal saline for gavage;retardant group was injected with testicular mesenchyme TGF-β1;Jingui Shenqi Pills group was givenJingui Shenqi Decoction for gavage. Blood was taken through caudal vein. Immunohistochemical, radioimmunoassay and electron microscopy observation were used to detect the expressions of TGF-β1, CYP19, sex hormone level, sperm density and motility rate and testicular morphological changes.Results Compared with normal group, the expression of TGF-β1 in the model group increased and CYP19 decreased (P<0.05). Compared with model group, the expression of TGF-β1 decreased and the expression of CYP19 increased (P<0.05). Compared with normal group, the number of sperm quantity was small and paramorphia in model group (P<0.05). Compared with model group, T, E2, sperm density and motility rate increased significantly inJingui Shenqi Pills group and retardant group (P<0.05). There was no statistical significance among indexes in retardant group andJingui Shenqi Pills group.ConclusionJingui Shenqi Pills can improve sperm quality and sex hormone level of model rats with kidney-yang-deficiency, which mechanism is probably realized by inhibiting the expression of TGF-β1R and promoting the expression of CYP19 in testis to effect development and proliferation of sperm.

Objective To explore the feasibility of endothelial function of dorsal artery of foot in patients with type 2 diabetes(T2DM) by high frequency ultrasound combined with warm bath test.MethodsThirty-five patients with T2DM and thirty normal people were collected,all subjects were examined by high frequency ultrasound.Diameter of brachial artery in baseline and after reactive hyperaemia were detected;Diameter of dorsal artery of foot in baseline and after the foot immersed in 40℃ warm water for 5 minutes were acquired.Flow mediated dilatation of dorsalis pedis artery(FMDDPA) and flow mediated dilatation of brachial artery(FMDBA) were calculated and compared.Multiple stepwise regression analysis was used to examine the correlation between FMDDPA and hemoglobin A1c(HbAlc). Results The FMDDPA and FMDBA were decreased in patients with T2DM (P<0.01).The FMDDPA and FMDBA were correlated significantly(r=0.864,P<0.01).In stepwise regression analysis,HbA1c is the most affecting factor for FMDDPA (R2=0.321,P<0.01).Conclusions Ultrasound combined with warm bath test can be used to detect the change of endothelial function of dorsal artery of foot in patients with T2DM,which have a certain clinical application value in endothelial function of terminal limb artery.

Purpose: It is well known that congenital diaphragmatic hernia (CDH) in infants impacts pulmonary function rehabilitation after surgery. However, the risk factors of postoperative pulmonary function are still unclear. In this research, we analyzed the potential risk factors of postoperative pulmonary function in CDH patients in order to improve the clinical management of CDH patients. Methods: Thirty-three cases CDH infants followed were enrolled from November 2016 to September 2018. Clinical data were reviewed. Tidal breathing pulmonary function testing was performed after surgery. Correlation between pulmonary function and clinical characteristics was evaluated using multivariate analysis of variance. Results: Pulmonary dysfunction was detected in 87.9% patients (29 of 33). The defect size was found to be significantly larger in patients with obstructed and mixed ventilatory disorders (P = 0.001). Diagnosis of gestational age (GA) was also significantly earlier compared to restrictive ventilatory disorders (P = 0.001). Larger defect size, and earlier prenatal diagnosis of GA were detected in severe obstructive ventilatory disorders (P = 0.007, P = 0.001, retrospectively). Conclusion Most patients had various degrees of pulmonary dysfunction after surgery. Patients with larger defect size and earlier diagnosis time might be vulnerable to severe obstructive and mixed ventilatory disorders.

Objective:To investigate the molecular genetic etiology of two fetuses with short rib-polydactyly syndrome type Ⅲ (SRPS Ⅲ).Methods:Next-generation sequencing (NGS) was used to detect 226 known genes related to inherited skeletal dysplasia in two fetuses with SRPS Ⅲ diagnosed in the First Affiliated Hospital of Zhengzhou University in August 2015 and June 2020. Suspect pathological variants were verified in the pedigree members using Sanger sequencing. The prenatal genetic diagnosis of the high-risk fetus in pedigree one was conducted to identify the confirmed pathogenic variation.Results:The homozygous mutation of DYNC2H1 gene c.5881A>G(p.Lys1961Glu) was identified in the proband in pedigree one, and the parents were the carriers. The proband in pedigree two carried compound heterozygous mutations in the DYNC2H1 gene with c.10606C>T(p.Arg3536*) inherited from the father and c.8954T>G(p.Val2985Gly) from the mother. Autosomal recessive inheritance was confirmed in both pedigrees. Mutations of c.5881A>G(p.Lys1961Glu) and c.8954T>G(p.Val2985Gly) in the DYNC2H1 gene were likely pathogenic variants and had not been reported before. The prenatal diagnosis did not identify the DYNC2H1 gene c.5881A>G(p.Lys1961Glu) mutation in the fetus (Ⅱ-7) in pedigree one, which was confirmed by the umbilical cord blood sample after birth. Conclusion:DYNC2H1 gene mutation underlies the fetal skeletal dysplasia in the two pedigrees.

OBJECTIVE: To explore the genetic basis for a child featuring congenital insensitivity to pain (CIP). METHODS: Targeted capture and next generation sequencing (NGS) was carried out for the proband. Suspected pathogenic variants were confirmed by Sanger sequencing of the proband and his parents. RESULTS: The proband was found to harbor compound heterozygous variants of SCN9A gene, namely c.1598delA (p.N533Ifs*31) and c.295_296delCGinsAT (p.R99I), which were respectively inherited from his father and mother. Both variants were predicted to be pathogenic, and neither was reported previously. CONCLUSION The compound heterozygous variants of the SCN9A gene probably underlay the CIP in this child. Above finding has enabled genetic counseling for this family.
Channelopathies ; Child ; High-Throughput Nucleotide Sequencing ; Humans ; Mutation ; NAV1.7 Voltage-Gated Sodium Channel/genetics* ; Pain Insensitivity, Congenital/genetics*

Objective: To establish a method for detection of human antibodies against monkeypox virus. Mothds: The enzyme linked immunosorbent assay (ELISA) plates were coasted with two monkeypox virus peptides from B21R protein, to establish an indirect ELISA for detecting monkeypox virus IgG antibody. The healthy individuals serum samples, monkeypox virus infected patient serum samples and other virus infected patient sera samples were applied to evaluate specificity of the peptides antigen. The reaction conditions were optimized. Results: Synthesized two peptides from monkeypox virus BR21R protein did not cross react obviously with healthy person serum and other virus infected serum. It was shown that the reaction condition was best with sera dilution at 1∶50 when two combined peptides were coated at 100 ng /well, and second-antibody was diluted at 1∶20 000. At this condition the cut off value of IgG antibody in serum samples for ELISA were A450 reading of 0.393. The detected results of two serum samples collected from the monkeypox patient in Sierra Leone were strongly positive, the titers of IgG antibody in two sera were both 1∶6 400. Conclusions The indirect ELISA for detection of monkeypox virus infection was established preliminarily which provided useful tools for epidemiological study and diagnosis.

Objective To investigate the efficacy of arthroscopic synovectomy on refractory knee arthritis complicated with popliteal cyst.Methods Patients diagnosed as rheumatoid arthritis (RA) or spondyloarthritis (SPA) with refractory knee arthritis who underwent knee arthroscopic synovectomy in our hospital from 2010 to 2017 were enrolled,including 20 patients (16 RA,4 SpA) with popliteal cyst.Clinical data,RA disease activity score (DAS28),SpA back pain score,etc,were collected to evaluate the efficacy of knee surgery.Results Erythrocyte sedimentation rate (ESR) [58(17,79)mm / 1h vs.19(9,30)mm/1h,P＜ 0.001],C reactive protein (CRP) [3.72(0.92,8.14) mg/L vs.0.85(0.10,3.08) mg/L,P＜0.001],rheumatoid factor [64.6(20.2,193.3) vs.20.5(10.0,58.4),P＜0.001],DAS28 score(4.67±1.25 vs.2.81±1.23,P＜0.001),knee joint discomfort score [5(4,6) vs.2(1,3),P＜0.001] and the volume of knee joint effusion by ultrasound (P＜0.05) in 95 RA patients were significantly decreased compared to those before operation.ESR [27(12,54)mm/1h vs.20 (16,28) mm/1 h,P＜0.001],CRP [3.27(1.06,6.95) mg/L vs.1.41(0.34,3.03)mg/L,P＜0.001],knee discomfort score [2(0,5) vs.1(0,3),P＜0.05],back pain visual analogue score (VAS) [5(4,5) vs.2(1,3),P＜0.001],and the volume of knee joint effusion by ultrasound (P＜0.001) in 58 SpA patients were significantly lower than those before the operation.The rate [16.84％(16/95) vs.6.32％(6/95),P=0.023] and grading (P=0.007) of popliteal cyst in RA were decreased after the operation.No statistically difference was observed in the rate [6.90％ (4/58) vs.5.17％(3/58),P=0.697] of popliteal cyst in patients with SpA,yet with a trend of decrease in 4 patients.Conclusion This study provide evidence that knee arthroscopic synovectomy has a good effect for refractory knee arthritis,which can reduce disease activity,improve joint symptoms and decrease the grading of popliteal cyst.

Objective: To generate monkeypox virus specific monoclonal antibodies for further establishing monkeypox virus immunofluorescence assay. Methods: Monkeypox virus A29 protein, vaccinia ortholog A27 protein and cowpox ortholog 162 protein were expressed in E. coli BL21 to screen antibodies. Synthetic monkeypox virus A29_{17 ~ 49} polypeptide was used to immune BALB/c mice. Monkeypox virus monoclonal antibodies were generated through fusion, cloning and screening techniques. Indirect ELISA was performed to test antibodies specificity and subtype. Results: A29, A27 and 162 proteins were highly expressed in E. coli and detected by Western blot. The three his-tagged proteins were purified using His-Bind affinity chromatography column. The purity of the proteins was all more than 90%. And 8 strains monkeypox virus specific monoclonal antibodies were screened by the three purified proteins. Two mAbs of 8 were IgG3 subtype and the rest were IgG1 subtype. Conclusions Eight strains of monkeypox virus specific monoclonal antibodies were generated, they can be used to further establish monkeypox virus immune immunofluorescence assay.

OBJECTIVE: To explore the genetic etiology for a Chinese pedigree affected with Meckel syndrome. METHODS: A pedigree with a history of three consecutive adverse pregnancies which presented at the First Affiliated Hospital of Zhengzhou University on August 31, 2017 was selected as the study subject. Clinical data of the pedigree were collected. High-throughput sequencing was carried out to screen for variants of ciliopathy-related genes in the third fetus following induced abortion, and candidate variant was verified by Sanger sequencing. RESULTS: The first pregnancy of the couple had ended as spontaneous abortion, whilst the fetus of the second pregnancy was suspected for having ciliopathy, though no genetic testing was carried out following elected abortion. The fetus of the third pregnancy was suspected for having ciliopathy, and high-throughput sequencing and Sanger sequencing had shown that the fetus had harbored compound heterozygous variants of the TMEM67 gene, including c.978+1G>A from the father and c.1288G>C (p.D430H) from the mother. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the c.978+1G>A was classified as a pathogenic variant (PVS1+PM2_Supporting+PP5), whilst the newly discovered c.1288G>C (p.D430H) was classified as a likely pathogenic variant (PM2_Supporting+PM3+PM5+PP3). CONCLUSION The c.978+1G>A and c.1288G>C (p.D430H) compound heterozygous variants of the TMEM67 gene probably underlay the three consecutive adverse pregnancies suspected for ciliopathy in this pedigree. The discovery of c.1288G>C (p.D430H) has also expanded the mutational spectrum of the TMEM67 gene.
Female ; Pregnancy ; Humans ; Pedigree ; East Asian People ; Ciliary Motility Disorders/genetics* ; Ciliopathies ; Abortion, Spontaneous ; Membrane Proteins/genetics*