High-dose omega-3 polyunsaturated fatty acid supplementation might be more superior than low-dose for major depressive disorder in early therapy period: a network meta-analysis.

BACKGROUND: The application of n-3 Polyunsaturated Fatty Acids (n-3 PUFAs) supplementation for major depressive disorder (MDD) has been widely discussed in recent years, but its efficacy and application are still controversial. This network meta-analysis was conducted to compare the efficacy of different dosages of n-3 PUFAs on MDD patients in the early period of treatment. METHODS: Randomized controlled trials (RCTs) exploring the efficacy of n-3 PUFA supplementation for patients with MDD were retrieved from the databases of Pubmed, Embase and the Cochrane Library. RCTs comparing the efficacy of n-3 PUFA for adult (≥18 years) MDD patients without comorbidity were eligible for our study. The score of depressive symptoms in early therapy period of the treatment (≤9 weeks) was extracted. Standardized mean deviations (SMDs) of all the sores from the eligible RCTs were synthesized in a pairwise meta-analysis in frequentist framework and a random-effects network meta-analysis in Bayesian framework for the overall and subgroups (high- and low-dose) efficacy of n-3 PUFAs. RESULTS: A total of 910 MDD patients in 10 trials with 3 adjuvant therapy strategies (high-dose n-3 PUFAs, low-dose n-3 PUFAs and placebo) were included. Results of pairwise meta-analysis showed that n-3 PUFAs were superior to placebo (SMD: 1.243 ± 0.596; 95% CI: 0.060 ~ 2.414). Results of the network meta-analysis showed that both the high (SMD: 0.908 ± 0.331; 95% CI: 0.262 ~ 1.581) and the low-dose (SMD: 0.601 ± 0.286; 95% CI: 0.034 ~ 1.18) n-3 PUFAs were superior to placebo, and the efficacy of high-dose n-3 PUFAs is superior to that of low-dose. CONCLUSIONS: High-dose n-3 PUFAs supplementation might be more superior than low-dose in the early therapy period for MDD. More head-to-head clinical trials need to be carried out to provide more direct comparison and enhance the evidence of the efficacy of n-3PUFAs for MDD.

Macromolecular crowding favors the fibrillization of ß2-microglobulin by accelerating the nucleation step and inhibiting fibril disassembly.

Hemodialysis-associated amyloidosis (HAA) involves the fibrillization of ß2-microglobulin (ß2M) and occurs in crowded physiological environments. However, how macromolecular crowding affects amyloid formation of ß2M remains elusive. Here we study the effects of macromolecular crowding on amyloid formation and fibril disassembly of wild-type human ß2M and its pathogenic mutant ΔN6. At strongly acidic pH2.5, the presence of a strong crowding agent (Ficoll 70 or dextran 70) not only dramatically accelerates the fibrillization of both wild-type ß2M and its ΔN6 variant by reducing the lag time to a large extent, indicating the acceleration of the nucleation phase, but also remarkably increases the amount of ß2M fibrils. At weakly acidic pH6.2, such an enhancing effect of macromolecular crowding on fibril formation is only observed for pathogenic mutant ΔN6, but not for wild-type ß2M which does not form amyloid fibrils in the absence and presence of a crowding agent. Thus, we propose that the monomers of ß2M form the nuclei, which is enhanced by macromolecular crowding, followed by the step of fibril elongation. Furthermore, at physiological pH, macromolecular crowding remarkably inhibits ß2M fibril disassembly by decreasing rate constants corresponding to fast and slow stages of fibril disaggregation. Our data demonstrate that macromolecular crowding favors the fibrillization of ß2M by accelerating the nucleation step and inhibiting fibril disassembly. Our findings provide clear evidence for the pathology of HAA that macromolecular crowding should be taken into account.

[Effects of co-expression plasmid of human tPA and VEGF165 on the proliferation of vascular cells and fibrinolysis activity].

OBJECTIVE: To observe the expression of the co-expression plasmid of tissue-plasminogen activator (tPA) and vascular endothelia growth factor165 (VEGF165) in vascular smooth muscle cells (VSMC) and to study the effect of expressing products on the proliferation of VEC and VSMC and fibrinolysis activity. METHODS: The co-expression plasmid of tPA and VEGF165 (pBudCE4.1/tPA-VEGF165) was transfected into VSMC with the lipofection. The expression of tPA and VEGF165 at mRNA level was detected by RT-PCR and the protein level expression was detected by enzyme linked immunosorbent assay (ELISA). The fibrinolysis activity of culture medium of VSMC transfected tPA and VEGF165 genes was detected by fibrin plate technique. The VEC and VSMC were cultured with culture medium of VSMC transfected tPA and VEGF165 genes. And the proliferation of VEC and VSMC was evaluated with monoterazolium (MTT) and flow cytometry (FCM). RESULTS: The expression of tPA and VEGF165 at mRNA and protein levels in the transfected VSMC was demonstrated by RT-PCR and ELISA, respectively. The VSMC culture medium of transfected genes possessed evidently fibrinolysis activity. The expression products of tPA and VEGF165 in the VSMC had an evident effect on the VEC proliferation. But it had not an effect on the VSMC proliferation. CONCLUSION: The eukaryotic co-expression plasmid of tPA and VEGF165 can be expressed in transfected VSMCs. The expression products have an obvious biological activity. The present study lay the foundation for future making use of tPA and VEGF165 to prevent and treat vascular stenosis in transplanted heart.

[To study the effects of local co-transfection vascular endothelial growth factor165 and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia after operation injury artery in rabbits].

OBJECTIVE: To observe the effects of local co-transfection vascular endothelial growth factor165 (VEGF165) and tissue-type plasminogen activator genes on inhibiting intimal hyperplasia and restenosis in rabbits artery after operation injury and possible mechanisms. METHODS: Micrology operation injury was used to establish the model of intimal injury of right external iliac artery in rabbits. To select 120 male New Zealand rabbits and were randomly divided into 3 groups (n = 40, in each group): Group A (physiological brine control group), Group B (pBudCE4.1 group), Group C (pBudCE4.1/VEGF165-tPA group). The vas-wall of micrology operation injury were infused respectively physiological brine, pBudCE4.1 and pBudCE4.1/VEGF165-tPA transfection solution by micro-injector. Each group were divided into 5 subgroups (n = 8, in each subgroup) randomly according to the sacrifice times (2 d, 1 week, 2 week, 4 week and 8 week after operation). The injured vascular specimen were harvested for pathology test, electric microscopy study, reverse transcription-PCR examining and immunochemistry detecting. RESULTS: The intimal area and narrow ratio of vases in Group C at every time point after operation were significantly lessened than that in Group A and Group B (P < 0.01). The narrow ratio of vases in Group C at 8 week after operation were decreased respectively by 57.9% and 59.0% than that in Group A and B. The expression of VEGF165 mRNA in Group C were increased significantly than that in Group A and B at every time point after operation (P < 0.01), the expression reached the peak at 1 week and continued to 4 week after operation. Immunohistochemical identified that tPA positive cell in Group C were significantly increased than that in Group A and B (P < 0.01) at every time point after operation. CONCLUSION: Local co-transfection VEGF165 and tPA genes could restrain intimal hyperplasia and restenosis of vas, which lay a foundation for future multi-gene therapy of vascular intimal hyperplasia.

[Therapeutic effects of DNA vaccines in a murine model of Mycobacterium tuberculosis infection].

OBJECTIVE: To study the effects and mechanisms of DNA vaccines encoding Mycobacterium tuberculosis antigens on murine Mycobacterium tuberculosis infection. METHODS: C57BL/J6 mice infected with Mycobacterium tuberculosis were treated with normal saline (NS), pCDNA3.1, psIL-12, pcD85B, pcDMPT64, pcD85B + pcDMPT64, and pcD85B + psIL-12 respectively. The numbers of viable bacteria in the lung and the spleen were counted. The levels of IFN-gamma, IL-4 and TNF-alpha of spleen lymphocytes stimulated with PPD were detected with ELISA. Lungs and spleens were prepared for pathological analysis. RESULTS: The pcD85B group (6.99 +/- 0.40 in lung, 5.17 +/- 0.33 in spleen), the psIL-12 group (7.41 +/- 0.50 in lung, 5.31 +/- 0.21 in spleen)and the pcD85B + psIL-12 group (7.64 +/- 0.28 in lung, 5.49 +/- 0.31 in spleen) showed significantly reduced number of colony forming units (lg(-1) CFU/g, x +/- s) in lungs and spleens compared with the control mice (5.76 +/- 0.16 in saline, 5.88 +/- 0.21 in pCDNA3.1), but the difference between the pcD85B group and the pcD85B + psIL-12 group was not significant. pcD85B vaccination induced high levels of IFN-gamma and TNF-alpha. No change of IL-4 was found in all groups. The pathological changes in lungs of the pcD85B group were localized, while those in the control group were extensive. There was no significant changes in the spleen of all groups. CONCLUSIONS: Ag85B DNA vaccination had immunotherapeutic effects, which were associated with a switch to Th1 response and enhanced production of cytokines TNF-alpha and INF-gamma. psIL-12 alone showed therapeutic effect, but it didn't enhance the therapeutic effect of single Ag85B DNA vaccination.

[Construction of co-expression plasmid containing tPA and VEGF165 genes and its expression in VSMCs].

OBJECTIVE: To construct the eukaryotic co-expression plasmid of tissue-plasminogen activator (tPA) genes and vascular endothelia growth factor165 (VEGF165) and observe its expression in vascular smooth muscle cells (VSMCs). METHODS: The tPA and VEGF165 genes were cloned from hominal heart tissue with RT-PCR, and then the tPA and VEGF165 genes were cloned into eukaryotic expression plasmid pBudCE4.1 to construct the eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165. The pBudCE4.1/tPA-VEGF165 was transfected into VSMCs via lipofection mediation. The expression levels of tPA and VEGF165 mRNAs in the transformed VSMCs were detected by RT-PCR and the expression levels of tPA and VEGF165 proteins were detected by ELISA. RESULTS: The size of RT-PCR product of tPA and VEGF165 genes was 1.9 kb and 576 bp respectively. Restrictive enzyme digestion analysis showed that recombinant co-expression plasmid pBudCE4.1/tPA-VEGF165 had been constructed successfully. The expression of tPA and VEGF165 at mRNA and protein levels in the transformed VSMCs and cells culture supernatant were detected respectively by RT-PCR and ELISA. CONCLUSION: The recombinant eukaryotic co-expression plasmid pBudCE4.1/tPA-VEGF165 has been successfully constructed. The tPA and VEGF165 are expressed in transformed VSMCs.

[Construction and expression of the eukaryotic coexpression plasmid containing Ag85B gene of Mycobacterium tuberculosis and IL-12 gene].

AIM: To construct an eukaryotic coexpression plasmid containing Mycobacterium tuberculosis (MTB) Ag85B and IL-12 genes. METHODS: MTB Ag85B gene and IL-12 gene were cloned into pBudCE4.1 which has multiple promoters to construct recombinant plasmid pBud85B-IL12. The recombinant plasmid was transfected into COS-7 cells and the expression of target genes was assessed by RT-PCR and ELISA. RESULTS: The expressions of Ag85B and IL-12 could be detected in COS-7 cells. CONCLUSION: The recombinant plasmid pBud85B-IL12 was constructed and expressed successfully, which lays the foundation for further development of DNA vaccine against tuberculosis.

[A study of the protective effect of the DNA vaccine encoding tubercle antigen 85B with MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To explore the protective effect of the DNA vaccine encoding Mycobacterium tuberculosis Ag85B with MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: Fifty four C57BL/6 mice were randomized into six groups and subjected to the following treatments respectively: intramuscularly immunized with PBS, pcDNA3.1, BCG, pcDNA/Ag85B, pcDNA/MPT64 and pcDNA/Ag85B + pcDNA/MPT64 on three occasions at 3-week intervals. The BCG group received a single subcutaneous injection of 1 x 10(6) CFU BCG. The mice were challenged with 10(6) CFU H(37)Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed, and the number of CFU in organs and histopathologic changes were determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: The number of bacterial colonies in the lungs and spleens were lg(-1) (7.854 +/- 0.003) CFU/g and lg(-1) (7.190 +/- 0.016) CFU/g in PBS group, lg(-1) (7.700 +/- 0.016) CFU/g and lg(-1) (7.072 +/- 0.068) CFU/g in pcDNA3.1 group, lg(-1) (6.449 +/- 0.002) CFU/g and lg(-1) (5.436 +/- 0.042) CFU/g in BCG group, lg(-1) (7.370 +/- 0.002) CFU/g and lg(-1) (6.430 +/- 0.009) CFU/g in pcDNA/Ag85B group, lg(-1) (7.547 +/- 0.003) CFU/g and lg(-1) (6.784 +/- 0.002) CFU/g in pcDNA/MPT64 group, and lg(-1) (6.918 +/- 0.002) CFU/g and lg(-1) (6.079 +/- 0.004) CFU/g in pcDNA/Ag85B + pcDNA/MPT64 group respectively, which showed significant decrease at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/Ag85B group was higher than that of the other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B + pcDNA/MPT64 group was higher than that of the other groups (P < 0.05). No IL-4 was detected in all groups. The pulmonary histopathological changes were observed 6 weeks later following challenge with Mycobacterium tuberculosis H(37)Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B group was characterized by serofibrous inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in the lung. The survival time of BCG vaccinated mice after challenge with Mycobacterium tuberculosis H(37)Rv was longer than that of the other groups. The survival time of pcDNA/Ag85B + pcDNA/MPT64 group was longer than that of other DNA vaccine groups. CONCLUSION: The protective effect of BCG was more significant than the other groups, while the effect of pcDNA/Ag85B + pcDNA/MPT64 was better than other DNA vaccines.

[The estimation of protective efficacy of the fusion gene vaccine encoding tubercle antigen 85B and MPT64 in mice challenged with Mycobacterium tuberculosis].

OBJECTIVE: To evaluate the protective efficacy of the fusion DNA vaccine (AM) encoding tubercle Ag85B and MPT64 in mice infected with Mycobacterium tuberculosis. METHODS: C57BL/6 mice were intramuscularly immunized with the DNA vaccines. The mice were challenged with 10(6) CFU H37Rv via lateral tail vein 35 days later after the third immunization for DNA vaccine groups and 100 days later for BCG vaccinated group. The mice in vaccinated groups and control groups were sacrificed 42 days later following challenge. The lungs and spleens were removed respectively, and the number of CFU in organs and histopathologic changes was determined. The antibody level, IFN-gamma, IL-4 and the survival time in all of the mice were evaluated. RESULTS: Antibody titer of pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). The level of IFN-gamma produced by spleen lymphocytes and spleen lymphocyte proliferation from BCG group, pcDNA/Ag85B, pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group was higher than that of other groups (P < 0.05). No IL-4 was found in all groups. The number of bacterial colonies in the lungs and spleens was significantly decreased at 6th week postchallenge in all the vaccinated groups (P < 0.05), especially in BCG group (P < 0.01). The pulmonary histopathological changes were observed 6 weeks later following challenge with M. tuberculosis H37Rv. In PBS and pcDNA3.1 groups, the lesion was characterized by seroplastic inflammatory infiltration and lung tissue necrosis, in BCG group by granulomas and numerous macrophages, lymphocytes and a few epithelioid cells. The lesion in pcDNA/Ag85B groups was characterized by seroplastic inflammatory infiltration and a few macrophages, in pcDNA/Ag85B + pcDNA/MPT64 group and pcDNA/AM group, by granulomas, numerous macrophages and lymphocytes. The lesion in spleen was different from the lung and characterized by proliferative lymphocytes and inflammatory infiltration. The results in spleen were similar to those in lung. The survival time of BCG vaccinated mice after challenge with M. tuberculosis H37Rv was longer than that of other groups. The survival time of AM group was longer than that of other DNA vaccine groups. CONCLUSION: The pcDNA/AM can improve the protective efficacy in immunized mice against M. tuberculosis.

[Preparation and application of recombinant Mycobacterium tuberculosis CFP10-ESAT-6 fusion protein].

OBJECTIVE: To prepare the recombinant CFP10-ESAT-6 fusion protein, and to study its immunological characteristics, and its potential for serodiagnosis of tuberculosis. METHODS: The lhp-ESAT-6 fusion gene was amplified by Gene SOEing, and then cloned into pQE30 plasmid. The recombinant CFP10-ESAT-6 fusion protein was expressed and purified. Its antigenicity was confirmed by Western blot. Animal models infected with M. tuberculosis H(37)Rv strain and M. bovis BCG respectively were made to evaluate the potential value of the fusion protein in the serodiagnosis of tuberculosis. RESULTS: The sequence of recombinant plasmid pQE30-CFP10-ESAT-6 was identical to the predicted sequence. The recombinant protein (rCFP10-ESAT-6), about 26 000, existed in the cytoplasm of DH5alpha in soluble form and represented 40% of the total bacterial protein. The purity and concentration of the final product was 98% and 1.2 g/L, respectively. Western blot showed that the rCFP10-ESAT-6 had good immunoreactivity with sera from patients with active tuberculosis and rabbits immunized with CFP10 and ESAT-6 respectively. The positive cutoff value was A(490) plus 2 standard deviation from negative guinea pig sera detected by ELISA. Serological reactivity to rCFP10-ESAT-6 was observed in 11 of the serum samples from guinea pigs with tuberculosis and 1 of sera from guinea pigs infected with BCG, while the serological reactivity to PPD was observed in 11 of sera from guinea pigs with tuberculosis and in 11 of sera from guinea pigs infected with BCG. CONCLUSIONS: The rCFP10-ESAT-6 fusion protein was highly expressed in soluble form in E. coli. It had antigenicity of both CFP10 and ESAT-6, and could be used to differentiate infection with M. tuberculosis H(37)Rv strain from immunization with M. bovis BCG. The study provided experimental data for potential application of rCFP10-ESAT-6 in the diagnosis of tuberculosis.

[Immunogenicity of DNA vaccine encoding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64].

AIM: To study the immunogenicity of a DNA vaccine encoding fusion protein of Mycobacterium tuberculosis Ag85B and MPT64. METHODS: C57BL/6 mice were intramuscularly injected respectively with PBS(A group), plasmid vector (B group), pcDNA/Ag85B(C group), pcDNA/MPT64(D group), and pcDNA/Ag85B MPT64 (E group) for three times with 2 week intervals. The mice were sacrificed 4 weeks after the final immunization. The total specific antibody levels were detected by ELISA and simultaneously spleen lymphocyte proliferation response to PPD and IFN-gamma level in culture supernatant of the PPD stimulated spleen lymphocytes was measured by MTT colorimetry and ELISA, respectively. RESULTS: The Ag85B, MPT64 and fusion gene Ag85B MPT64 DNA vaccines could induce anti-PPD antibody production as well as antigen specific lymphocyte proliferation and high levels of IFN-gamma in immunized mice. CONCLUSION: The Ag85B MPT64 fusion gene can induce specific cellular immunity and humoral immunity in immunized mice.

[Construction of the prokaryotic expression vector of MTB lhp gene and its expression].

AIM: To construct prokaryotic expression vector carrying lhp gene and express it in E.coli. METHODS: The MTB lhp gene was amplified by PCR and then cloned into plasmid pQE30. After sequencing, the gene was cloned into plasmid pET32a(+) to construct recombinant prokaryotic expression vectors pQE30-CFP10 and pET32a(+)-CFP10. RESULTS: After transformation of the E.coli and induction with 1 mmol/L of IPTG, no additional protein was expressed in pQE30-CFP10 system, but recombinant target protein with M(r) 20 000 or so was expressed in pET32a(+)-CFP10 system, and the expressed protein was maximum when induced with IPTG for 4 h. The expressed protein existed in cytoplasm in soluble form and amounted to 38% of total protein of E.coli. Western blot analysis showed that the protein had good antigenicity. The purity of the protein purified through the Ni-NTA resin reached 93%. CONCLUSION: The prokaryotic expression vector pET32a(+)-CFP10 was constructed successfully and the rCFP10 protein was obtained, which laid the foundation for application of the rCFP10.