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The extracellular domain (p75ECD) of p75 neurotrophin receptor (p75NTR) antagonizes Aβ neurotoxicity and promotes Aβ clearance in Alzheimer's disease (AD). The impaired shedding of p75ECD is a key pathological process in AD, but its regulatory mechanism is largely unknown. This study was designed to investigate the presence and alterations of naturally-occurring autoantibodies against p75ECD (p75ECD-NAbs) in AD patients and their effects on AD pathology. We found that the cerebrospinal fluid (CSF) level of p75ECD-NAbs was increased in AD, and negatively associated with the CSF levels of p75ECD. Transgenic AD mice actively immunized with p75ECD showed a lower level of p75ECD and more severe AD pathology in the brain, as well as worse cognitive functions than the control groups, which were immunized with Re-p75ECD (the reverse sequence of p75ECD) and phosphate-buffered saline, respectively. These findings demonstrate the impact of p75ECD-NAbs on p75NTR/p75ECD imbalance, providing a novel insight into the role of autoimmunity and p75NTR in AD.
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Mice , Animals , Alzheimer Disease/pathology , Receptor, Nerve Growth Factor , Amyloid beta-Peptides , Autoantibodies , Mice, TransgenicABSTRACT
Brucellosis has become a global zoonotic disease, seriously endangering the health of people all over the world. Vaccination is an effective strategy for protection against Brucella infection in livestock in developed countries. However, current vaccines are pathogenic to humans and pregnant animals, which limits their use. Therefore, it is very important to improve the safety and immune protection of Brucella vaccine. In this study, different bioinformatics approaches were carried out to predict the physicochemical properties, T/B epitope, and tertiary structure of Omp2b and Omp31. Then, these two proteins were sequentially linked, and the Cytotoxic T lymphocyte associated antigen-4 (CTLA-4) variable region was fused to the N-terminal of the epitope sequence. In addition, molecular docking was performed to show that the structure of the fusion protein vaccine had strong affinity with B7 (B7-1, B7-2). This study showed that the designed vaccine containing CTLA-4 had high potency against Brucella, which could provide a reference for the future development of efficient brucellosis vaccines.
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Objective:To evaluate the immunogenicity of a novel influenza virus mRNA vaccine based on conserved antigens delivered by lipopolyplex (LPP) platform in a mouse model.Methods:Four copies of genes coding for extracellular domain of matrix 2 protein (M2e) and nucleoprotein (NP) of influenza A virus were synthetized after codon optimization. The fusion antigens were transcribed in vitro and delivered by LPP platform, named as LPP-4M2eNP. Expression of M2e and NP in eukaryotic cells was detected by immunofluorescence assay (IFA). BALB/c mice were inoculated intramuscularly twice with 10 μg or 30 μg LPP-4M2eNP vaccine at an interval of four weeks. Antibody response was detected by ELISA and cellular-mediated immunity (CMI) was detected by enzyme-linked immunospot assay (ELISPOT). Results:IFA showed that NP and M2e were expressed correctly in eukaryotic cells. Single dose immunization could induce significant antigen (NP, M2e)-specific CMI and antigen (NP, M2e)-specific antibody response was induced in mice with Th1 type bias after boost immunization. Moreover, NP-specific CMI was increased significantly after the second immunization, while no significant change in M2e-specific CMI was observed.Conclusions:Stronger CMI was triggered in mice by single dose of LPP-4M2eNP vaccine. Furthermore, robust humoral and cellular immune responses were induced after boost immunization. This study suggested that LPP-4M2eNP vaccine, which based on conserved antigen of influenza A and delivered by LPP platform, had great potential for development and application.
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Alzheimer's disease (AD) is a neurodegenerative disease characterized by cognitive impairments with progressive loss of memory and behavioral disorder.Up to now,there is no effective therapy or drug to cure AD.Recent studies have shown p75 neurotrophin receptor (p75NTR) plays a critical role in the pathogenesis of AD,while the extracellular domain of p75 neurotrophin receptor (p75ECD) has neuroprotective effect and can attenuate the development and progression of AD.Therefore,p75ECD is a research-hotspot for prevention and treatment of AD.Here,recent studies are reviewed to learn about the advances of p75ECD in the prevention and therapy of AD and provide references for getting novel methods and drugs to treat AD.
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Objective To investigate serum p75 neurotrophin receptor-extracellular domain (p75NTR-ECD) level in patients with chronic cerebral hypoperfusion-vascular cognitive impairment (CCH-VCI) and its relationship with tumor necrosis factor α (TNF-α), interleukin (IL)-1β and IL-6. Methods The clinical data of patients with CCH-VCI (n＝34) were collected from Changhai Hospital, Naval Medical University (Second Military Medical University) from Aug. to Dec. 2018. Enzyme linked immunosorbent assay was applied for detection of serum levels of p75NTR-ECD, TNF-α, IL-1β and IL-6; and the results were then compared with those of ischemic stroke participants (n＝34) and healthy controls (n＝36), who were all in the same age range. Spearman correlation analysis was used to analyze the relationship between serum p75NTR-ECD level and the above-mentioned inflammatory factors in CCH-VCI patients. Results The serum p75NTR-ECD level in the CCH-VCI group was significantly higher than those in the healthy control group and the ischemic stroke group (544.36 [440.88, 628.50] pg/mL vs 276.49 [262.59, 313.87] pg/mL and 366.87 [337.09, 450.43] pg/mL, U＝87.500 and 335.500, both P0.05). The serum levels of TNF-α, IL-1β and IL-6 were 196.02 (141.20, 280.35) pg/mL, 68.23 (60.79, 91.94) pg/mL and 51.04 (40.24, 65.26) pg/mL in the CCH-VCI group, respectively, and 218.67 (143.76, 281.28) pg/mL, 76.87 (59.10, 99.91) pg/mL and 64.45 (43.13, 86.76) pg/mL in the ischemic stroke group, respectively, which were all significantly higher than those in the healthy control group (73.71 [56.94, 79.81] pg/mL, 42.98 [34.52, 51.34] pg/mL and 14.97 [11.76, 21.19] pg/mL, respectively; U＝ 31.000 and 4.000, 106.000 and 132.000, and 48.000 and 13.000; all P0.05). Serum p75NTR-ECD level in the CCH-VCI patients was correlated with TNF-α level (r＝0.391, P＝0.022), but not with IL-1β or IL-6 levels (r＝0.032 and 0.164, P＝ 0.855 and 0.355). Conclusion Serum p75NTR level may be related to inflammatory factors (TNF-α) after chronic cerebral hypoperfusion, and they may jointly participate in the pathogenesis of CCH-VCI.
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@#This study is performed to analyze the anti-liver fibrosis effect of the fusion protein of human serum albumin and extracellular domain of transforming growth factor beta type II receptor(eTGFBR2)in vivo to looking for the more stable anti-liver fibrosis drug. The mice model of liver fibrosis was constructed by CCl4 induction and the following groups are included in the study: the control group, CCl4 model group, the positive control group, eTGFBR2 treatment group, HSA-eTGFBR2 treatment group, and HSA group. Hematoxylin eosin staining, serum liver function index detection, and western blot are used to identify the anti-liver fibrosis activities. The results showed that: (1)CCl4 caused liver structure disorder, hepatocellular necrosis, collagen fibers proliferation, and induced liver fibrosis at last; (2)HSA-eTGFBR2 and its monomer drug improved the symptoms of liver fibrosis significantly, as well as reduced the damage of liver cells and collagen deposition, and recovered the liver basic structure to normal. Both of HSA-eTGFBR2 and its monomer drug improved liver function and reduced the expression level of liver fibrosis marker α-SMA and COL I. Moreover, the anti-liver fibrosis effect of the fusion protein is comparable to the monomer drug. In contrast, the albumin had no effect on therapeutic effect; (3)Reducing the injection frequency of HSA-eTGFBR2 achieved the comparable effects to the monomer drug with the normal injection frequency. In summary, the fusion protein HSA-eTGFBR2 has good anti-liver fibrosis effect. In addition, reducing the injection frequency of the fusion protein could also achieve the comparable treatment with the monomer drug, indicating that the fusion protein is stable and has longer half-lives and then a relatively positive application prospect in future.
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Objective To study the clinical significance for expression of serum matrix metalloproteinase and HER-2 extracellular domain in primary breast cancer.Methods Two hundred and forty two early breast cancer patients were enrolled as research group who met the criteria in Peking University Shenzhen Hospital from May 2015 to October 2017.They were divided into HER-2 positive breast cancer group (n =53) and HER-2 negative breast cancer group (n =189) according to HER-2 status.At the came time,non breast cancer women were recruited in outpatient as control group.The concentrations of serum MMP-2,MMP-9 and HER-2 ECD were detected by ELISA,and the differences of concentration in each grup were compared.Measurement data were expressed by mean and standard deviation,t test was used between two groups of parameters.A single factor analysis of variance was used among the multiple groups of parameters,and LSD test was used in the comparison between every two groups.Results The serum levels of MMP-2,MMP-9 and HER-2 ECD were (12.07 ± 1.23) ng/ml,(25.20 ±3.53) ng/ml and (10.42 ± 6.08) ng/ml in research group,HER-2 positive group were (13.43 ± 4.63) ng/ml,(25.24 ± 2.12) ng/ml and (13.25 ± 3.42) ng/ml,HER-2 negative group were (10.55 ± 3.72) ng/ml,(23.16 ±3.21) ng/ml and (6.33 ±4.11) ng/ml.The serum levels of MMP-2,MMP-9 and HER-2 ECD in the control group were (9.03 ± 1.15) ng/ml,(21.15 ±2.12) ng/ml and (4.71 ± 1.93) ng/ml.There were significant differences in MMP-2,MMP-9 and HER-2 ECD concentration between research group and control group (all P ＜ 0.0001).There was significant difference between HER-2 positive group and HER-2 negative group,HER-2 positive group and control group in the HER-2 ECD (P ＜ 0.0001).There were significant difference between control group and HER-2 positive group,control group and HER-2 negative group in the MMP-2 and MMP-9(P ＜ 0.000 1).In the HER-2 positive group,there was significant difference between HER-2 ECD positive and HER-2 ECD negative subgroup of MMP-2 and MMP-9(P ＜ 0.000 1).Conclusions In early breast cancer,HER-2 positive is an important condition for HER-2 ECD positive,and associated with abnormal activation of MMP.Combined detection of serum HER-2 ECD,MMP-2 and MMP-9 contributes to the determination of HER-2 status in tissues.
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Objective To express and purify the glycoprotein extracellular domain (Ex-GP) of Rabies virus strain CTN in soluble form with high efficiency.Methods A recombinant expression plasmid containing the gene encoding the Ex-GP was constructed.Various expression conditions were screened to obtain an optimum prokaryotic expression system for Ex-GP in soluble form.The expressed target protein was purified using affinity chromatography and gel filtration chromatography.Results The target protein Ex-GP with high antigenicity was efficiently expressed in soluble form by using the recombinant PBCX expression system and effectively purified by using affinity and gel filtration chromatography.Conclusion The soluble form of Ex-GP is successfully expressed and purified in a simple and convenient way.This study paves the way for further researches on the biological functions of rabies virus glycoprotein,the pathogenic mechanism of rabies and the development of diagnostic reagent and vaccines for rabies virus.
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Background & objectives: The proto-oncogene HER2/neu has been extensively studied in breast cancer patients. Serum levels of HER2/neu by ELISA in breast cancer patients were compared with tissue HER2/neu expression and with other clinicopathological parameters with the aim to investigate whether the serum assay could replace the established tests (IHC/FISH) for HER-2 status. Methods: Blood and Tru-cut biopsy samples were collected for determining HER2/neu status in 64 breast cancer patients. The tissue specimens were processed routinely and immunohistochemistry (IHC) for HER2/ER/PR (oestrogen/progesterone receptors) performed. Fluorescence in-situ hybridization (FISH) was performed on all HER2/neu 2 positive cases. Sixty age matched healthy females and females with benign breast disease were taken as controls for ELISA. Results: Of the 64 breast cancer cases, 25 (39.1%) had elevated serum HER2/neu levels accompanied with increased tissue expression of HER2/neu receptors. On IHC, HER2/neu score was 3+ in 24 (37.5%) cases, 2+ in three (4.6%), 1+ in 18 (28.1%); while 19 cases (29.7%) showed no HER2/neu expression. Of the three 2+ cases on IHC, two showed amplification on FISH. Twenty one (32.8%) patients were ER positive and 17 (26.6%) were PR positive. There was a significant correlation (P<0.001) of serum HER2 concentration with tumour size, lymph node involvement, stage of disease and histological grade. Serum HER2/neu levels showed a negative correlation with ER status (P=0.047) but no correlation with PR status. Interpretation & conclusions: The results suggest that elevated serum HER2 level was associated with a clinicopathological aggressive phenotype of breast carcinoma and was related to tissue HER2 overexpression. Therefore, serum HER2 may be useful for monitoring the course of the disease and response to treatment.
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Objective:In the present study,Bac-to-Bac baculovirus expression system was used to obtain recombinant human Cosmc extracellular domain protein,which can lay the foundation for the research about the structure and function of Cosmc protein in vitro,and simultaneously provide ideas for the research of O-glycosylation and related diseases. Methods: The Cosmc extracellular domain ( Cosmc-ED) gene was cloned into a transfer vector pFastBac1 to form the recombinant donor plasmid pFastBac1-Cosmc ED, which was transformed into competent cells DH10Bac. By using blue-white selection and PCR analysis,we could obtain recombinant shuttle vector rBacmid-Cosmc ED. Then, the recombinant gene DNA of rBacmid-Cosmc ED was used to transfect Sf-9 mediated by cationic lipid formulation,and the recombinant baculovirus bacmid was obtained,which was further used to infect the serum-free cell Sf-9 to express Cosmc-ED in the supernatant. Then the protein of interest was detected by SDS-PAGE and Western blot and purified with Ni-NTA affinity column. Results:SDS-PAGE and Western blot analysis showed a specific band about 33 kD,consistent with the interest protein. Mass spectrometry results further prove that the protein was Cosmc extracellular domain protein. Conclusion: Human Cosmc-ED protein can be successfully expressed in Sf-9 insect cells and laid basis for subsequent studies.
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Trypanosomosis or surra is caused by the haemoflagellate parasite, Trypanosoma evansi and is an important disease of animals, including domestic and wild herbivores and carnivores, in tropical countries. The invariant surface glycoproteins (ISGs) are blood stream stage specific and are uniformly distributed over the entire surface of the trypanosomes. In the present study, the extracellular domain (ED) region of ISG-75 from T. evansi, consisting of 1320 nt, encoding a polypeptide of 440 amino acids, has been heterologously expressed in Escherichia coli. Further, the immunoreactivity of recombinant ISG-75 (rISG-75) was characterized in immunoblot and ELISA using T. evansi hyper immune sera raised in experimental animals. The protein was found immunoreactive when compared with a panel of antigens (VSG RoTat 1.2 and whole cell lysate) using bovine serum samples from field. The diagnostic potential of rISG-75 was evaluated in ELISA with large number of bovine field serum samples. The optimum sensitivity and specificity were 98.47 and 99.1, respectively. The present finding showed that the expressed protein has potential use in the serodiagnosis of trypanosomosis.
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Animals , Base Sequence , Cattle , Cattle Diseases/diagnosis , DNA Primers , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay/methods , Membrane Glycoproteins/diagnosis , Polymerase Chain Reaction , Protozoan Proteins/diagnosis , Recombinant Proteins/diagnosis , Sensitivity and Specificity , Serologic Tests/methods , Trypanosomiasis/diagnosis , Trypanosomiasis/veterinaryABSTRACT
Circulating tumor markers have been paid more attention in the application of the treatment for breast cancer, the level of which has extended from protein to gene, including traditional tumor markers, HER-2 extracellular domain, circulating tumor cells, circulating tumor DNA (ctDNA), circulating RNA (ctRNA) and so on. As “liquid detection”, the detection of circulating tumor markers with real-time dynamic, easy operation, good reproducibility and other advantages are widely used in aiding early diagnosis, determining prognosis, prospectively predicting response or resistance to speciifc therapies, surveillance after primary surgery, and monitoring therapy in patients with advanced disease, The further study of circulating tumor markers may contribute to patient’s individual treatment.
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AlM:To construct recombination eukaryotic expression plasmid of human thyrotropin receptor extracellular domain encapsulated with cationic liposomes. METHODS:We amplified the target gene of shuttle vector PHMCMVTSHR289, conjugated the target gene and eukaryotic expression plasmid pcDNA3. 1 +, and accredited whether pcDNA3. 1+/TSHR289 was connected or not by enzymatic digestion and sequencing. Cationic liposomes encapsulated the recombination plasmid pcDNA3. 1+/TSHR289. RESULTS: Recombination plasmid pcDNA3. 1+/TSHR289 digested with enzyme Hindlll and the fragment through 0. 8% gel electrophoresis showed 512bp strip. Recombination plasmid pcDNA3. 1+/TSHR289 were found synonymous mutation through forward ( AAC to AAT ) and reverse sequencing ( GCG to GCT) . The volume ratio of cationic liposomes and recombinant plasmid was 3:1. CONCLUSlON: lt is successful to construct the recombination plasmid pcDNA3. 1+/TSHR289 by accredit it through enzymatic digestion and sequencing.
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PURPOSE: The extracellular domain (ECD) of the HER-2/neu oncoprotein, whose molecular weight is the range from the 95 kD to 105 kD, is shed into the blood from the cell surface via, proteolysis by a metalloprotease. A monoclonal antibody immunoassay has been developed for measuring the circulating concentrations of serum HER-2/neu ECD (following serum HER-2/neu). Serum HER-2/neu has been reported to be correlated with an increased tumor volume in those patients suffering with breast cancer. We measured the serum CA15-3 level, which is a surrogate marker of the tumor burden, we analyzed the correlation of the serum CA15-3 with the serum HER-2/neu and we analyzed the association of both markers with the clinical outcomes. METHODS: The sera for the analysis of both HER-2/neu and CA15-3 were obtained from 99 healthy Korean women, 66 primary breast cancer patients and 43 metastatic breast caner patients. The serum HER-2/neu level was measured quantitatively with using an ADVIA Centaur(R) automated immunoassay analyzer (Bayer Health Care LLC, Diagnostics Division, Tarrytown, New York, USA) and the CA 15-3 level was measured via radioimmunoassay. RESULTS: The serum HER-2/neu level was increased 23 metastatic cancer patients (53%). On the analysis of the correlation of serum HER-2/neu and CA15-3, the correlation coefficient (r) was 0.8072. Thus a positive serum HER-2/neu test in breast cancer patients was highly associated with the CA15-3 level for assessing whether metastasis was present or not. For the relationship between primary breast cancer and metastatic breast cancer, the former was classified as the control group and the latter as the patient group. The results of the Receiver Operation Characteristic (ROC) curve for serum HER-2/neu and CA15-3 showed no statistically significant differences (p=0.176) but the diagnostic efficacy of the serum HER-2/neu test was measured more exactly than that of CA15-3 and CA15-3 a tool for measuring a tumor marker for the diagnosis of whether metastasis was present or not. CONCLUSION: Serum HER-2/neu is a significant independent predictive prognostic factor for metastatic breast cancer patients. In view of the results we have achieved so far the serum HER-2/neu level in metastatic breast cancer patients may play an important roll as an independent tumor marker.
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Female , Humans , Biomarkers , Breast , Breast Neoplasms , Delivery of Health Care , Immunoassay , Molecular Weight , Neoplasm Metastasis , New York , Proteolysis , Stress, Psychological , Tumor BurdenABSTRACT
Objective To eukaryotically exp ress B7.1 extracellular domian and investigate its biological activities. Methods Recombinant expression vector pDisplay/B7.1(V+C) wa s transfected into Hela cells by electroporation method. The expression of B7.1( V+C) was identified by immunohistochemistry and in situ hybridization, respectiv ely. In vitro T lymphocyte activation activity was detected by 3H-TdR incoporation method and the aroused cytotoxicity after mixed culture of pDispla y/B7.1(V+C) transfected Hela cell-T cell was observed by MTT method. R esults B7.1(V+C) was expressed successfully on the surface of Hela c ells. The expressed B7.1(V+C) was able to activate T lymphocytes in the pres ence of anti-CD3 monoclonal antibody. Cytotoxicity could be observed when pDisp lay/B7.1(V+C) transfected Hela cells were mixed and co-cultured with T lymphocy tes. Conclusion Eukaryotically expressed B7.1(V+C) main tains its T lymphocyte costimulatory activity, and B7.1(V+C) can be adopted to construct bifunctional molecules for tumor immunotherapy.
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Objective: To study the cellular immune response and the anti-tumor effects induced by intramuscular DNA immu- nization with HER2/neu extracellular domain gene. Methods: HEH2/neu oncogene extracelltilar domain gene was isolated and inserted to pCDNA3 expression vector. The cellular immune response and its anti-tumor effects were analyzed after intramuscular DNA immunization. Results: A specific cytotoxic activity by spleen cells from HER2/neu ECD gene immunized mice was found in vitro. Tumor growth was inhibited after tumor challenge in vivo in immunized mice. Conclusion: Specific cellular immune re- sponse and anti-tumor effect were elicited by HEB2/neu extracellular domain gene immunization.
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AIM: To clone the extracellular domain of N-cadherin cDNA, and to observe the antigenicity of the expressed protein. METHODS: Total RNA was extracted from CD34+ cells separated from fetal liver and bone marrow cells. The extracellular domain of N-cad cDNA was amplified with RT-PCR and inserted into a vector pOPE101-215. The recombinant pOPE-N-cad was expressed with IPTG induction. Then, mice were immunized with the protein. RESULTS: The extracellular domain of N-cad cDNA from CD34+ cells was identified by DNA sequencing. The recombinant pOPE-N-cad in host XL1-blue expressed a 70 kD protein after induced with IPTG, and anti-N-cad antibody was detected in serum of the immunized mice after 5 times injection of the recombinant N-cad protein. CONCLUSION: CD34+ cells bore N-cad gene and the recombinant protein of the extracellular domain of N-cad cDNA shows good immunogenicity.
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Objective To construct the prokaryotic expression vector of mouse CD25 extracellular domain and to express it in E coli.Methods Total RNA was isolated from splenocytes of Balb/c mice.The CD25 extracellular domain gene was amplified by RT-PCR and cloned into the PET-32a vector.A positive recombinant,PET-32a-CD25e,was identified by enzyme cleaving and sequencing before its expression in E.coli,and transferred into E.coli BL21(DE3) plysS for its expression.After purification with Ni+ resin and renaturation in vitro,a relative molecular mass(Mr) of the interesting protein was detected by SDS-PAGE and Wes-tern blotting.Effect of the purified interesting protein on the proliferation of splenocytes from T cell vaccine-immunized syngeneic mice was detected by MTT assay.Results The cloned CD25 extracellular domain gene was identified to be functional by sequencing and expression.The purified interesting protein could significantly induce the proliferation and IL-4 secretion of splenocytes from T cell vaccine-immunized mice in vitro.Conclusion Mouse CD25 extracellular domain gene can be successfully expressed in prokaryotic cells with biological activity,which lays a foundation for further relative studies.