Associations between brain imaging-derived phenotypes and cognitive functions.

We aimed to evaluate the potential causal relationship between brain imaging-derived phenotypes and cognitive functions via Mendelian randomization analyses. Genetic instruments for 470 brain imaging-derived phenotypes were selected from a genome-wide association study based on the UK Biobank (n = 33,224). Statistics for cognitive functions were obtained from the genome-wide association study based on the UK Biobank. We used the inverse variance weighted Mendelian randomization method to investigate the associations between brain imaging-derived phenotypes and cognitive functions, and reverse Mendelian randomization analyses were performed for significant brain imaging-derived phenotypes to examine the reverse causation for the identified associations. We identified three brain imaging-derived phenotypes to be associated with verbal-numerical reasoning, including cortical surface area of the left fusiform gyrus (beta, 0.18 [95% confidence interval, 0.11 to 0.25], P = 4.74 × 10-7), cortical surface area of the right superior temporal gyrus (beta, 0.25 [95% confidence interval, 0.15 to 0.35], P = 6.30 × 10-7), and orientation dispersion in the left superior longitudinal fasciculus (beta, 0.14 [95% confidence interval, 0.09 to 0.20], P = 8.37 × 10-7). The reverse Mendelian randomization analysis indicated that verbal-numerical reasoning had no effect on these three brain imaging-derived phenotypes. This Mendelian randomization study identified cortical surface area of the left fusiform gyrus, cortical surface area of the right superior temporal gyrus, and orientation dispersion in the left superior longitudinal fasciculus as predictors of verbal-numerical reasoning.

[Gene cloning, expression and binding activity of anti-CD86 scFv].

OBJECTIVE: To construct and express the single chain variable fragment (scFv) against human CD86 in CHO cells, and observe its biological functions of binding with antigen. METHODS: The V(H); and V(L); genes were cloned by RT-PCR from a murine hybridoma cell line 1D1, which produced the monoclonal antibody (mAb) against human CD86. The CD86-scFv gene was integrated into eukaryotic expression vector to construct pIREST2-EGFP/scFv. CHO cells were transfected by pIREST2-EGFP/scFv plasmid with Lipofectamine(TM); 2000 and then were selected by G418. We used IMAC affinity chromatography to purify CD86-scFv and quantified its concentration by BCA method. Then the identification of CD86-scFv binding to membrane CD86 was performed through competitive inhibition assay. In addition, the growth inhibition effect of CD86-scFv on Raji cells was detected via MTT assay. RESULTS: One cell line stably expressing CD86-scFv was obtained. The CD86-scFv could identify CD86 molecules on L929-CD86, Raji and Daudi cells, and the positive rates were 67.0%, 72.3% and 80.5%, respectively. CD86-scFv showed the competitive binding to murine parent antibody 1D1. Moreover, CD86-scFv inhibited the growth of Raji cells and the inhibition rate was 28.3% 72 h after 20 µg/mL CD86-scFv was added into Raji cells. CONCLUSION: CD86-scFv has been successfully expressed in CHO cells (named SA-IV) and the antibody shows a good biological function of recognizing CD86 and inhibiting the growth of Raji cells.

[Expression and antigen binding activity of anti-human CD86 diabody].

AIM: To construct a recombinant eukaryotic expression vector of anti-human CD86 diabody gene and express anti-CD86 diabody through Chinese hamster ovary (CHO) cells, then analyze the capability of the diabody to recognize the tumor cells expressing CD86 and its biological effect. METHODS: The antibody heavy and light chain viable region gene (V(H); and V(L);) were cloned from hybridoma cell 1D1 which secreted anti-human CD86 monoclonal antibody. Anti-human CD86 diabody gene V(H);-(GGGGS)-V(L); was constructed by SOE-PCR. Then we inserted it into eukaryotic expression vector to construct a recombinant vector pIRES2-EGFP/CD86-diabody. The recombinant vector was transfected into CHO cells with Lipofectamine(TM); 2000, and the cell clones secreting CD86 diabody were screened by G418. We used IMAC to purify CD86 diabody and quantified its concentration by BCA method. The capability of the diabody to recognize the CD86 expressed on Raji and Daudi was analyzed through flow cytometry. After Raji cells were treated with CD86 diabody for 72 h, its proliferation inhibiting effect was investigated by MTT assay. RESULTS: We have obtained one CHO cell line that stably secreted CD86 diabody. The concentration of CD86 diabody after purification was 5.24 mg/L. The positive rates of CD86 diabody to recognize Raji and Daudi were 77.2% and 70.6%, respectively. After CD86 diabody treatment for 72 h, the inhibition rate of Raji cells was 37%. CONCLUSION: The anti-human CD86 diabody which we obtained successfully could recognize CD86 expressed on tumor cells specifically, and inhibit the proliferation of these tumor cells effectively.

[Effect of CD80-scFv on recognition of CD80 molecule and proliferation of different tumor cells in vitro].

AIM: To prepare CD80-scFv in CHO cells and investigate its effect on the recognition and proliferation of different tumor cells. METHODS: The CD80-scFv was purified from culture supernatant without fetal calf serum (FCS) by IMAC affinity chromatography, and then bound to CD80 molecule on the Daudi, U251, A375 and 8266 cells detected by flow cytometry. Different concentrations of CD80-scFv were added in the training system of different tumor cells, and we analyzed the influence on the proliferation of these cells by MTT assay. With 8266 cells which expressed CD80 highly and were treated by mitomycin firstly as the APC, and human PBMC as the effect cells, we analyzed the influence of the CD80-scFv on the proliferation of PBMC by blocking the co-stimulatory signal. RESULTS: The concentration of CD80-scFv purified from FCS was about 6.67 mg/L and the binding rate of CD80-scFv with Daudi, U251, A375, 8266 and U266 were 96.8%, 39.6%, 20.5%, 99.9% and 3.8%, respectively. CD80-scFv suppressed the proliferation of Daudi and 8266 cells which expressed CD80 highly, and the inhibition rate of Daudi and 8266 cells cultured with CD80-scFv (the final concentration was 25 µg/mL) was 24.04% and 24.16%, respectively. Additionally, the antibody suppressed the proliferation of PBMC by blocking the co-stimulatory signal mediated by CD80-CD28. CONCLUSION: CD80-scFv can recognize CD80 molecule and suppress the proliferation of tumor cells which express CD80 highly.

[Changes in the expression of T lymphocyte subsets in ageing mice induced by D-galactose].

AIM: To explore the changes and significance of spleen T cell subsets in ageing mice induced by D-galactose. METHODS: Ageing mice model was successfully established by 100 g/L D-galactose. The content of IFN-γ and IL-4 in serum was measured by ELISA. T cell subsets were detected by Immunofluorescence technique and flow cytometry. RESULTS: The content of IFN-γ and IL-4 in the serum was decreased of model group (P<0.01). The naive T cell related molecule, CD45RA was decreased(P<0.05). T cell activation-related molecule, CD25 was decreased(P<0.05). The Foxp3 in CD4(+);CD25(+); T cell was increased(P<0.01). CONCLUSION: In spleen of the ageing mice, the percentage of naive and active T cell are decreased, but The percentage of CD4(+); Tr subset is increased.

[Changes and effects of significant membrane molecules on thymic T cell of aging mice induced by D-galactose].

AIM: To establish subacute aging mice model by D-galactose and to explore the changes and effects of significant membrane molecules on thymic T cell. METHODS: Female Kunming mice of 8 weeks old were injected with D-galactose of 12.5 mL/(kg.d) by subcutaneous in scruff for 42 days. The animals' living conditions and biological behaviors were observed everyday.SOD activities and MDA content of serum were measured to determine whether the aging model was successfully established.On the basis of successfully establishing aging model, detect the significant membrane molecules of thymic T cell by Immunofluorescence technique and Flow Cytometer. RESULTS: During the 42 days, gradually, the model mice showed bending body, loose skin, slow action and so on.The activities of SOD in the serum were significantly decreased(P<0.01), and the content of MDA in the serum was significantly increased(P<0.01). The thymic naive T cell significant molecule, CD45RA was decreased(P<0.05). T cell activation-related molecules, CD28 and CD25 were both decreased(P<0.05), and PD-1 was significantly increased(P<0.01). The memory T cell significant molecule, CD196 was increased, but was not significantly compared to the control mice. CONCLUSION: The D-galactose subacute aging mice model was successfully established.The naive and active T cell were decreased and the memory T cell was increased in the thymic of the aging.

[The changes of spleen B cells of D-galactose-induced aging mice].

AIM: This paper is to analyze the changes of important membrane type molecules of the spleen B cells and their significance on the basis of biology identification by establishing subacute aging mice model with D-galactose. METHODS: Health Kunming mice are injected with 100 ng/L D-galactose, 0.25 mL/20 g, once per day, for 42 days consecutively, into their back necks. The animals' living conditions and biological behaviors are observed everyday and the dynamic changes of their weight are measured regularly. The biology identification of aging model mice is conducted by means of measuring the SOD viability and malondialdehyde (MDA) concentration in serum; the analysis of the activation- and memory-related important membrane type molecules of spleen cells is carried out with immune fluorescence and flow cytometry; the analysis of the concentration of IL-4 in serum is conducted by ELISA. RESULTS: Building a successful subactue aging mice model.The results of immune fluorescence and flow cytometry showed that CD20(+); was 56.8%, CD40(+); was 43.6%, CD20(+);CD45RA(+);B was 14.04%, CD40(+);CD45RA(+);B was 35.4%, CD20(+);CD86(+);B was 2.25%, CD40(+);CD86(+);B was 4.38%, CD20(+);CD196(+);B was 10.68%, and CD40(+);CD196(+);B was 10.98%. The results of ELISA showed that the average level of IL-4 in serum was 7.93 ng/L. The statistical analysis showed that the expressions of CD20, CD40, CD45RA, and CD86 of the spleen cells of the model control group and the average level of IL-4 in serum were lower than the normal control group(P<0.05)while CD196 was higher than the normal control group(P<0.01). CONCLUSION: In the body's aging process, the expressions of the activation- and memory-related important membrane type molecules of spleen cells change, activated cells reduce, memory cells increase, and the expression of IL-4 in serum drop, resulting in the immune system function disorder.

[Construction of human CXCR3B gene transfected cell line, identification and study of its biological function].

AIM: To construct recombinant human CXCR3B gene expression vector and obtain L929-CXCR3B gene transfected cell line for stably expressing human CXCR3B. METHODS: Human CXCR3B gene of full length was amplified by PCR from the plasmid pMD19-T/huCXCR3A. Then, it was inserted into eukaryotic expression vector pIRES2-EGFP to construct recombinant vector pIRES2-EGFP/huCXCR3B. The recombinant vector was transfected into L929 cells with Lipofectamine(TM); 2000, and cell clones stably expressing human CXCR3B molecule were screened by G418.We used FCM and RT-PCR to verify expression of CXCR3B from protein level and gene level. The ability of proliferation of L929-huCXCR3B under the circumstance of CXCR3B ligand called Mig was analyzed via MTT methods. RESULTS: We have constructed recombinant human CXCR3B gene expression vector and obtained L929-huCXCR3B gene transfected cell line which can stably express human CXCR3B molecule. The positive expression rate of CXCR3B on L929-huCXCR3B cells was 93&. The result of MTT assay showed that the proliferation of L929-huCXCR3B cells were inhibited when the cells were cocultured with Mig after 24 h, 48 h and 72 h, and the inhibition ratio were 41.44&, 44.01& and 24.80& respectively. CONCLUSION: Construction of L929-huCXCR3B cell line has laid a good foundation on research of the CXCR3B signal pathway and preparation of the CXCR3B monoclonal antibody.

The negative co-signaling molecule b7-h4 is expressed by human bone marrow-derived mesenchymal stem cells and mediates its T-cell modulatory activity.

Though experimental evidence shows that human bone marrow-derived mesenchymal stem cells (hBMSCs) are able to suppress T-cell activation and proliferation, the precise mechanisms are still not completely understood. Here, we investigated the role of the negative costimulatory molecule B7-H4 in the immunosuppressive effect of hBMSCs on T-cell activation. We showed that B7-H4 expresses abundantly on hBMSCs assessed by reverse transcription, immunofluorescence staining, and flow cytometric analysis. Further studies demonstrated that B7-H4 expressed on hBMSCs inhibits T-cell activation and proliferation via induction of cell cycle arrest and inhibition of NF-kappaB nuclear translocation. Blocking B7-H4 would decrease the secretion of transforming growth factor-beta1 (TGF-beta1) in the supernatant of activated T cells co-cultured with hBMSCs. Addition of neutralizing antibodies against B7-H4 significantly attenuated the inhibitory effects of hBMSCs on T-cells. Thus, our study established the novel role of B7-H4 molecule in the suppressive effect of hBMSCs on T-cell activation and proliferation. Taken together, these results highlight the complex role of hBMSCs in regulating the immune response, asserting the possibility of their therapeutic application in transplantation, the treatment of graft-versus-host disease (GVHD), and autoimmune diseases.

Cloning, purification and bioactivity assay of human CD28 single-chain antibody in Escherichia coli.

Engineered single chain antibodies have become a powerful source of immunotherapy against a wide range of diseases. Here, we present the generation of human CD28 single-chain antibody gene (CD28-ScFv), which contained variable fragments of heavy chain and light chain (VH and VL) of the anti-CD28 antibody, and a linking peptide (Gly4Ser)3 inserted in the middle of VH and VL. The fused gene CD28-ScFv was successfully expressed in BL21 (DE3) cells and confirmed by western blotting assay. The molecular weight of CD28-ScFv was 43 kDa and the major fraction was expressed as an insoluble body. By dissolving the insoluble bodies, renaturing in vitro and purifying with a Ni-NTA affinity column, highly purified expression products of CD28-ScFv were obtained. This product could recognize and bind to CD28+ positive T cells. The proliferation capacity of peripheral blood T cells was increased by purified CD28-ScFv. In this study, we improved orthodox renaturing techniques by combining the dilution renaturation with phase gradient dialysis. With this new method, highly purified CD28-ScFv products were developed and biological activity of the products was similar to that of the mouse monoclonal anti-human CD28 antibody.

[Inhibitory and lethal effects of human-mouse chimeric antibody against CD80 on the growth of B lymphoma cell lines Raji and Daudi].

AIM: To study the inhibitory and lethal effects of human-mouse chimeric antibody against CD80 (named ch-4E5) on the growth of B lymphoma cell lines Daudi and Raji. METHODS: Immunofluorescence and flow cytometry were used to analyze the detection of membrane CD80 in Raji and Daudi by ch-4E5. After the co-culture of ch-4E5 with Raji and Daudi, respectively, at the final concentration of 10 mg/L, the expression of Fas and FasL was observated to be at the 0 h, 4 h, 10 h, 16 h, 24 h and 48 h by direct immunofluorescence and flow cytometry, and the blocking effect on cell growth of ch-4E5 was determined at 72 h by MTT assay. MTT assay was also used to study the ADCC effect with PBMC as effector cells and Raji and Daudi as target cells. The efficiency target ratio was 20:1. RESULTS: The combination rate between ch-4E5 and Raji and Daudi was 98.6% and 96.4%, respectively. After the co-culture of ch-4E5 with Raji and Daudi cells for 4 hs, the expression of FasL in Raji began to up-regulate. It reached the peak at 16 h and its positive rate was 16.8%. Compared with human IgG1 control group, it was increased obviously (P<0.01). The expression of Fas increased at 10 h, and then reached the top at 24 h. The combination rate was 15.6%. There was significant deviation compared with human IgG1 control group (P<0.01). Moreover, the expression of FasL and Fas on Daudi was also altered. The trend was similar to these on Raji, and the highest expression was 15.9% and 13.7%, respectively. The inhibitory rate was 34.60% and 32.64% respectively when ch-4E5 with Raji and Daudi had been co-cultured for 72 h (P<0.01). Furthermore, ch-4E5 could mediate the ADCC effect and the maximum killing rate was 55.61% and 54.42%, respectively(P<0.01). CONCLUSION: The human-mouse chimeric antibody against CD80 can inhibit the proliferation of B lymphoma cell lines Daudi and Raji cells through Fab and Fc sections in vitro.

[Dynamic changes of cytokines in G-CSF mobilized peripheral blood].

OBJECTIVE: To investigate the level and significance of interleukin-8 (IL-8), soluble intercellular adhesion molecule (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) in patients peripheral blood (PB) during mobilization for peripheral blood stem cells harvesting. METHODS: The levels of IL-8, sICAM-1 and sVCAM-1 in patients were dynamically assayed by ELISA during the mobilization procedure and the number of CD(34)(+) cell, white blood cell (WBC) and platelet (BPC) by flow cytometric analysis and hematometry respectively. Colony formation was assayed by using semisolid methycellulose culture. RESULTS: There was a significant increase in plasma levels of IL-8 and both adhesion molecules [IL-8 (247.4 +/- 84.2) microg/L (P < 0.01); sICAM-1 (530.3 +/- 286.1) microg/L (P = 0.002 7); sVCAM-1 (575.3 +/- 350.4) microg/L (P = 0.001 3)] during the mobilization process; furthermore, IL-8 and sVCAM-1 concentration in the patient's plasma was paralleled to the numbers of CD(34)(+) cell, CFU-GM, WBC and BPC (P < 0.001). CONCLUSION: The levels of IL-8, sICAM-1 and sVCAM-1 in the patient's plasma were correlated to the PB number of CD(34)(+) cells, CFU-GM, WBC and BPC during the mobilization process. It suggested that analysis of IL-8, and sVCAM-1 dynamic changes may serve as markers for CD(34)(+) cells.