Chemokine CCL24 promotes the growth and invasiveness of trophoblasts through ERK1/2 and PI3K signaling pathways in human early pregnancy.

Chemokine CCL24, acting through receptor CCR3, is a potent chemoattractant for eosinophil in allergic diseases and parasitic infections. We recently reported that CCL24 and CCR3 are co-expressed by trophoblasts in human early pregnant uterus. Here we prove with evidence that steroid hormones estradiol (E), progesterone (P), and human chorionic gonadotropin (hCG), as well as decidual stromal cells (DSCs) could regulate the expression of CCL24 and CCR3 of trophoblasts. We further investigate how trophoblast-derived CCL24 mediates the function of trophoblasts in vitro, and conclude that CCL24/CCR3 promotes the proliferation, viability and invasiveness of trophoblasts. In addition, analysis of the downstream signaling pathways of CCL24/CCR3 show that extracellular signal-regulated kinases (ERK1/2) and phosphoinositide 3-kinase (PI3K) pathways may contribute to the proliferation, viability and invasiveness of trophoblasts by activating intracellular molecules Ki67 and matrix metallopeptidase 9 (MMP9). However, we did not observe any inhibitory effect on trophoblasts when blocking c-Jun N-terminal kinase (JNK) or p38 pathways. In conclusion, our data suggests that trophoblast-derived CCL24 at the maternal-fetal interface promotes trophoblasts cell growth and invasiveness by ERK1/2 and PI3K pathways. Meanwhile, pregnancy-related hormones (P and hCG), as well as DSCs could up-regulate CCL24/CCR3 expression in trophoblasts, which may indirectly influence the biological functions of trophoblasts. Thus, our results provide a possible explanation for the growth and invasion of trophoblasts in human embryo implantation.

CPY27B1-related vitamin D deficiency contributes to the impaired function of CD4+T cells in Recurrent spontaneous miscarriage.

The study aimed to explore the relationship between the expression of cytochrome P450 family 27 subfamily B member 1 (CYP27B1), vitamin D, and impaired T cell subsets in recurrent spontaneous miscarriage (RSM). A Total of 779 healthy women of childbearing age and 1031 women with a history of RSM were involved in this study. The results of flow cytometry showed that the proportion of Tregs was higher in healthy women than in the women with RSM. For cytokines, the levels of interleukin-17 (IL-17) and interferon-gamma (IFN-γ) were significantly higher in RSM patients than in healthy women, while IL-10 was notably lower in RSM patients. Furthermore, compared to healthy individuals, RSM patients had lower levels of serum 25(OH)D detected by chemiluminescence. The frequency of Tregs was negatively correlated with 25(OH)D. Specifically, for every 10 ng/ml increase in 25(OH)D, the percentage of Tregs increased by 0.58 as calculated. IL-17 and IFN-γ were inversely correlated with 25(OH)D, while the serum interleukin-10 (IL-10) level was positively correlated with 25(OH)D. CYP27B1 was found to be expressed in both cytotrophoblast and extracellular villi trophoblast cells. However, reduced expression of CYP27B1 was observed in the placenta with RSM. Notably, the level of 25(OH)D increased in the supernatant of CYP27B1 knockdown BeWo compared to normal cells, while human chorionic gonadotropin (hCG) was significantly reduced. The hCG secretion of CYP27B1 KO BeWo cells was partially restored after 1,25(OH)2D3 supplementation. In addition, 1,25(OH)2D3 treatment could induce more CD4+ T cells to convert to Foxp3+iTreg, which in turn inhibited the secretion of IL-17, IFN-γ. In summary, this research unveiled a connection between reduced CYP27B1 and vitamin D deficiency in RSM. Our study underscores the potential benefits of vitamin D treatment supplementation in the context of RSM. However, it is important to note that further research is imperative to validate these observations.

Assessing progress towards achieving the transport dimension of the SDGs in China.

Transport is an important service industry in the national economy. Sustainable transport is central to sustainable development. Currently, investigating the sustainable development process and trade-offs in China's transport sector is urgent. In this study, 11 transport indicators were selected and constructed for the sustainable development goals (SDGs) under the UN indicator framework. The scores of each indicator were calculated, and spatiotemporal patterns and interactions were analyzed. The results revealed that China's transport infrastructure performed well in large transportation volumes and guaranteed traffic safety and strict land use control, with scores above 75. However, China's transport sector currently faces a challenge in using clean energy, and a more balanced development of bus ownership among the provinces is expected. The interaction analysis revealed three pairs of indicators with synergy (ρ > 0.5), but both the significant negative and positive relationships among the selected indicators accounted for approximately half, indicating the development of sustainable transport in China would move in zigzags. Road accessibility was an indicator interacting with most sustainable transport indicators. We suggest that more SDG indicators with indirect impacts should be included in future sustainable transport research.

Noise Emission Models of Electric Vehicles Considering Speed, Acceleration, and Motion State.

Electric vehicles, known for their low-noise emission, are popular and widespread in metropolises in China, and they provide an opportunity for a reduction in environmental noise from vehicles. To understand the noise from electric vehicles better, this study develops noise emission models considering speed, acceleration, and motion state. The model construction is based on the data collected from a pass-by noise measurement experiment in Guangzhou, China. The models describe a linear relationship between the noise level, the logarithm of speed, and the acceleration for multiple motion states (i.e., the constant-speed state, the acceleration state, and the deceleration state). From the spectrum analysis, the low-frequency noise is barely affected by the speed and acceleration, but the noise at a certain frequency is most sensitive to them. Compared to other models, the proposed ones have the highest accuracy and the greatest ability for extrapolation and generalization.

Deep Learning-Based Road Traffic Noise Annoyance Assessment.

With the development of urban road traffic, road noise pollution is becoming a public concern. Controlling and reducing the harm caused by traffic noise pollution have been the hot spots of traffic noise management research. The subjective annoyance level of traffic noise has become one of the most important measurements for evaluating road traffic pollution. There are subjective experimental methods and objective prediction methods to assess the annoyance level of traffic noise: the subjective experimental method usually uses social surveys or listening experiments in laboratories to directly assess the subjective annoyance level, which is highly reliable, but often requires a lot of time and effort. The objective method extracts acoustic features and predicts the annoyance level through model mapping. Combining the above two methods, this paper proposes a deep learning model-based objective annoyance evaluation method, which directly constructs the mapping between the noise and annoyance level based on the listening experimental results and realizes the rapid evaluation of the noise annoyance level. The experimental results show that this method has reduced the mean absolute error by 30% more than the regression algorithm and neural network, while its performance is insufficient in the annoyance interval where samples are lacking. To solve this problem, the algorithm adopts transfer learning to further improve the robustness with a 30% mean absolute error reduction and a 5% improvement in the correlation coefficient between the true results and predicted results. Although the model trained on college students' data has some limitations, it is still a useful attempt to apply deep learning to noise assessment.

Restorative effect of audio and visual elements in urban waterfront spaces.

Introduction: Urban waterfront spaces are often composed of built infrastructures and nature elements. Though citizens could take advantage of these public spaces to relax from daily work, its restorative potential has not been paid enough attention. In this study, the restorative effect and mechanism of different audio and visual elements in urban waterfront spaces was systematically studied. Methods: At the first stage, restorative potential of waterfront spaces was investigated and different elements with restorative effects were identified through an on-site survey, in which visual and auditory forms of environmental-nature, animal-nature, on-water human activities and on-shore human activities were identified. At the second stage, a series of laboratory experiments were conducted to explore the restorative function of the audio and visual elements. Results and discussion: It is found that the degree of artificiality of waterfront space was a crucial factor influencing the restoration level of the space, and higher artificiality level of waterfront space resulted in lower level of perceived restoration. However it was available by adding visual and audio elements to the scene to facilitate the restorative effect in waterfront spaces with high-level artificiality. The effects of adding visual and auditory elements on psychophysiological restoration were explored, and elements that should be recommended and restrained were discussed. Prospects: These findings would provide applicable suggestions for future design and rebuilding of urban waterfront spaces.

The effects of audio-visual perceptual characteristics on environmental health of pedestrian streets with traffic noise: A case study in Dalian, China.

The COVID-19 pandemic has affected city dwellers' physical and mental health and has raised concerns about the health of urban public spaces. This field investigation research in Dalian, China, examined the perceived audio-visual environment characteristics of urban pedestrian streets with traffic noise and their influences on the environmental health of the pedestrian streets. Five indicators reflecting psychological responses to environmental characteristics (willingness to walk, relaxation, safety, beauty, and comprehensive comfort) were used to measure environmental health of pedestrian streets with traffic noise. The results showed that safety was rated the highest, and willingness to walk was evaluated as the lowest among health evaluation indicators. The imageability and openness of the streetscape were associated with each health evaluation indicator. In contrast, the rhythm and continuity of the street buildings had a greater effect on willingness to walk than the other health indicators. There were negative correlations between L Aeq for traffic noise and health evaluations. Positive health evaluations were observed when L Aeq was less than 55 dBA. In contrast, soundscape indicators showed positive correlations with health evaluations, and acoustic comfort and noise annoyance, rather than sound preference and subjective loudness were associated with each health evaluation indicator. In terms of the combined audio-visual factors, acoustic comfort, the quantity of greening, annoyance, sky visibility, spatial scale, and building distance were examined as the determining factors affecting health evaluations, and 55.40% of the variance in health evaluations was explained by the soundscape and streetscape indicators. The findings provide references for better understanding the relationships between healthy experience and audio-visual perceptions. Moreover, they enable environmental health quality optimisation of pedestrian spaces considering audio-visual indicators and approaches in the post-epidemic era.

Sperm DNA fragmentation valued by SCSA and its correlation with conventional sperm parameters in male partner of recurrent spontaneous abortion couple.

The objective of this study is to evaluate the predictive value of sperm DNA fragmentation Index (DFI) in unexplained recurrent spontaneous abortion (RSA) and to investigate its correlation with conventional sperm parameters. Besides, we aimed to reveal the necessity of establishing a DFI clinical threshold of each laboratory for the prognostic diagnosis of RSA and establish our own DFI threshold. Semen samples were collected from male partners of RSA patients (n = 139) and healthy recent fathers (control, n = 200). DFI was tested using SCSA and conventional semen analysis was performed using an automatic semen analyzer. The DFI value and distribution were compared between the two groups using corresponding statistical software. The diagnostic threshold value was established by ROC curve. The correlation between DFI and the conventional semen parameters of the 139 cases was further analyzed using Student's t test and Mann-Whitney U test. Our result showed that DFI was significantly higher in RSA patients compared with normal donor controls. We established our own DFI threshold at 13.59%. There was only a weak partial correlation between DFI values and conventional sperm analysis parameters. Our present study suggested that DFI might be used as a valuable predictor for RSA independent of conventional sperm parameters. Additionally, we recommend that each laboratory should establish its own clinical DFI threshold for more precise prediction of RSA and we recommend that sperm DNA fragmentation test should be included in complete sperm quality assessment in addition to conventional semen analysis for RSA male partners.

Hydroxychloroquine reverses the drug resistance of leukemic K562/ADM cells by inhibiting autophagy.

Autophagy is an essential metabolic pathway mediated by lysosomal degradation, which is involved in scavenging and recycling senescent or damaged organelles and biological macromolecules in eukaryotic cells. The present study explored the association between the autophagic activity and chemotherapy resistance of leukaemia cells, and the possibility of using autophagy inhibitors to combat leukemic drug resistance. It was found that the levels of basic autophagy in multidrug­resistant leukaemia cells (K562/ADM) were significantly higher compared with sensitive cells (K562), and that Adriamycin (ADM) was capable of inducing autophagic activity in K562 and K562/ADM cells. K562 and K562/ADM cells were treated with a series of hydroxychloroquine (HCQ) concentrations to inhibit cellular autophagy and detect cell sensitivity to ADM. The results demonstrated that the sensitivity of K562 cells to ADM was mildly enhanced by HCQ, and that the sensitivity of K562/ADM cells to ADM was markedly strengthened by HCQ. In addition, more typical morphological changes associated with apoptosis emerged, and the ratio of Bax/Bcl­2 and activity of caspase­3 were markedly increased in K562/ADM cells treated with HCQ. Notably, the expression of mdr1 mRNA and P­glycoprotein (P­gp) in drug­resistant K562/ADM cells was upregulated along with increasing autophagic activity induced by ADM. Furthermore, HCQ significantly reduced the increase in P­gp expression by inhibiting autophagic activity. Collectively, these findings indicated that the inhibition of autophagy significantly promoted the sensitivity of K562/ADM cells to ADM by facilitating apoptosis. Furthermore, inhibition of autophagy attenuated the expression of P­gp; therefore, P­gp may be involved in autophagic regulation in drug­resistant cells.

Cisplatin inhibits the growth, migration and invasion of cervical cancer cells by down-regulating IL-17E/IL-17RB.

Interleukin (IL)-17E mainly produced by immune cells, is a distinct member of the IL-17 cytokine family, which has multifarious immunomodulatory activities. As a potent anticancer drug, cisplatin is commonly used against various types of solid tumors. The present study was performed to investigate whether cisplatin regulates the expression of IL-17E and it receptor IL-17RB, and the role of IL17E in cervical cancer cells in vitro. The expression of IL-17E and IL-17RB in cervical cancer cells was detected by flow cytometry and ELISA. The viability, apoptosis, migration and invasion of cervical cancer cells were analyzed by CCK8, Annexin V-7AAD apoptosis, transwell migration, wound healing, and matrigel invasion assays. Here, we found that cervical cancer cells co-expressed IL-17E and IL-17RB, especially HeLa and SiHa cells. Recombinant human IL-17E protein (rhIL-17E) enhanced the viability, migration and invasion of HeLa and SiHa cells, and blocking IL-17E with anti-human IL-17RE neutralizing antibody promoted the apoptosis of HeLa and SiHa cells. Cisplatin significantly down-regulated the expression of IL-17E and IL-17RB, and further reversed the regulatory effects of rhIL-17E on viability, apoptosis, migration and invasion of HeLa and SiHa cells. The results suggest that cisplatin inhibits the viability, migration, invasion, and promotes the apoptosis of cervical cancer cells possibly by down-regulating IL-17E/17RB signaling. Cisplatin may be the first choice for cervical cancer patients with abnormally high IL-17E expression.

Differences of basic and induced autophagic activity between K562 and K562/ADM cells.

Patients with acute myeloid leukemia (AML) often have a poor prognosis due to drug resistance, which is regarded as a tough problem during the period of clinical therapeutics. It has been reported that autophagy, an important event in various cellular processes, plays a crucial role in mediating drug-resistance to cancer cells. Our study attempts to comparatively investigate the differences of basic and induced autophagic activity between drug-sensitive and multidrug-resistant AML cells. The level of basic autophagy in K562/ADM cells was higher than that in K562 cells, which could be characterized by more cytosolic contents-packaged autophagic vacuoles in K562/ADM cells when compared to that in K562 cells. The observation of MDC staining showed that the fluorescent intensity of autophagosomes in K562/ADM cells was stronger than that in K562 cells. The expression of Beclin1 and the ratio of LC3-II to LC3-I were distinctly higher in K562/ADM cells, however, P62 protein was relatively lower in K562/ADM cells. Furthermore, we found that nutrient depletion could induce autophagic activity of both cell lines. However, autophagic activity of K562/ADM cells was always maintained at a higher level in contrast with K562 cells. ADM (Adriamycin) was also capable of inducing autophagic activity of K562 and K562/ADM cells, but the autophagic alteration in K562 cells appeared earlier. Taken together, our findings suggest that autophagy exerts an important effect on formation and maintenance of drug-resistance in AML cells.

Human thymic stromal lymphopoietin promotes the proliferation and invasion of cervical cancer cells by downregulating microRNA-132 expression.

Thymic stromal lymphopoietin (TSLP), produced by cervical cancer (CC) cells, promotes angiogenesis, and the recruitment and functional regulation of eosinophils. It has been reported that microRNA (miR)-132 is aberrantly decreased in CC tissues. However, the function and mechanism of TSLP on the biological behaviors of CC cells is largely unknown. The aim of the present study was to investigate the effect of TSLP on the expression of miR-132 and the proliferation and invasion in vitro of CC cell lines, namely, HeLa and SiHa cells. The transcrpitional level of miR-132 was analyzed using reverse transcription-quantitative polymerase chaon reaction. The proliferation, invasion, and the expression of proliferation and invasion-related molecules in HeLa and SiHa cells in vitro were evaluated using bromodeoxyuridine cell proliferation, Matrigel invasion assays, flow cytometry and ELISA, respectively. Here, it was revealed that recombinant human TSLP (rhTSLP) downregulated the expression levels of miR-132 in HeLa and SiHa cells, and by contrast, the neutralizing antibodies for TSLP or TSLP receptor (TSLPR) upregulated miR-132 expression levels in HeLa and SiHa cells. The overexpression of miR-132 resulted in a lowered proliferation and invasiveness, decreased levels of proliferation-associated molecules marker of proliferation Ki-67 and proliferating cell nuclear antigen, and the decreased production of matrix metalloproteinase (MMP)2 and MMP9 in HeLa and SiHa cells. Compared with the control group, there was a higher level of proliferation and invasion in HeLa and SiHa cells following stimulation with rhTSLP. However, these effects induced by rhTSLP were significantly impaired in HeLa and SiHa cells with miR-132 overexpression. The results of the present study indicated that TSLP produced by CC cells downregulated miR-132 expression, and stimulated the proliferation and invasion of CC cells, thereby further promoting the development of CC.

Tim-3 protects decidual stromal cells from toll-like receptor-mediated apoptosis and inflammatory reactions and promotes Th2 bias at the maternal-fetal interface.

Toll-like receptors (TLRs) are important in mediating immune responses against various pathogens during pregnancy. However, uncontrolled TLR-triggered inflammation will endanger normal pregnancy, resulting in pregnancy loss. Therefore, maintenance of a moderate inflammatory response is crucial for successful pregnancy under conditions of infection. Here, we demonstrated significantly lowered expression of T-cell immunoglobulin and mucin domain 3 (Tim-3) in miscarried decidual stromal cells (DSCs), indicating that Tim-3 might play important roles in maintaining successful pregnancies. Activation of TLR signaling induced pro-inflammatory cytokine production and apoptosis of DSCs, which was accompanied by up-regulated Tim-3 expression. Tim-3, in turn, protected DSCs from TLR-mediated apoptosis in an ERK1/2 pathway-dependent manner. In addition, Tim-3 inhibited TLR signaling-induced inflammatory cytokine production by DSCs through suppressing NF-κB activation. Tim-3 increased production of T helper 2 (Th2)-type cytokines by DSCs and reversed the inhibitory effect of LPS on Th2 cytokine generation by up-regulation of interferon regulatory factor 4 expression. Tim-3 blockade abolished the effect of Tim-3 on the inflammatory response to LPS stimulation. Thus, Tim-3 signaling could represent a "self-control" mechanism in TLR-triggered inflammation during pregnancy. These findings identify Tim-3 as a key regulator of DSCs and suggest its potential as a target for the treatment of spontaneous abortion.

Combination of CD4(+)CD25(+)CD127(-) regulatory T cells with MLC-BE and BE-Ab2: an efficient evaluation of the therapy of paternal lymphocyte induced immunization in unexplained recurrent spontaneous abortion patients.

The aim of this retrospective study was to compare the immune tolerance status of patients suffered from unexplained spontaneous abortion (URSA) before and after treatment with paternal lymphocyte induced immunization (PLII) four times, and its relationship to the pregnancy outcome. 168 URSA patients were included in the present study. Among 168 couples, 138 couples were conceived again, of whom 86 were successfully pregnant till 20 gestational weeks, 31 cases again failed in the first trimester, 21 cases were still under follow-up, another 30 cases still had not conceived. Both the level of one way mixed lymphocyte culture blocking efficiency (MLC-BE) and anti-idio blocking antibody (BE-Ab2) were markedly elevated in succeeded group after PLII. In contrast, although a significant increase could be observed in the failed group after treatment, the elevation of BE-Ab2 was much lower than that in successful group. PLII therapy significantly up-regulated the percentage of peripheral CD4(+)CD25(+)CD127(-) regulatory T cells (Tregs) in successfully pregnant women; however, there was no significant change of Tregs in pregnancy loss cases although receiving PLII therapy. These results suggested a positive correlation between higher frequency of Tregs and rate of successful pregnancies. The sensitivity and specificity of combination of Tregs with MLC-BE and BE-Ab2 were 81.8% and 81.3%, respectively. Therefore, the percentage of Tregs in peripheral blood may hopefully serve as a potential biomarker for monitoring the efficacy of therapy in URSA patients. Combination of Tregs with MLC-BE and BE-Ab2 may expect to better evaluate the efficacy of PLII in URSA patients.

Chemokine CCL17 induced by hypoxia promotes the proliferation of cervical cancer cell.

Cervical cancer is often associated with hypoxia and many kinds of chemokines. But the relationship and role of hypoxia and Chemokine (C-C motif) ligand 17 (CCL17) in cervical cancer are still unknown. Here, we found that CCL17 was high expressed in cervical cancer. HeLa and SiHa cells could secrete CCL17 in a time-dependent manner. Hypoxia increased expression of CCL17 receptor (CCR4) on HeLa and SiHa cells. Treatment with recombination human CCL17 (rhCCL17) led to an elevation of cell proliferation in HeLa and SiHa cells in a dose-dependent manner. In contrast, blocking CCL17 with anti-human CCL17 neutralizing antibody (α-CCL17) played an oppose effect. However, rhCCL17 had no effect on apoptosis in cervical cancer cells. Further analysis showed that hypoxia promoted the proliferation of HeLa and SiHa cells, and these effects could be reversed by α-CCL17. Stimulation with the inhibitor for c-Jun N-terminal kinase (JNK) or signal transducers and activator of transcription 5 (STAT5) signal pathway not only directly decreased the proliferation of HeLa and SiHa cells, but also abrogated the stimulatory effect of rhCCL17 on the proliferation of HeLa and SiHa cells. These results suggest that a high level of CCL17 in cervical cancer lesions is an important regulator in the proliferation of cervical cancer cells through JNK and STAT5 signaling pathways. In this process, hypoxia magnifies this effect by up-regulating CCR4 expression and strengthening the interaction of CCL17/CCR4.

Enhancement of humoral immunity to the hCG beta protein antigen by fusing a molecular adjuvant C3d3.

The vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical test demonstrating the feasibility of the approach in preventing pregnancy in women. Some individuals, however, did not response adequately despite employing highly immunogenic bacterial toxoids as carriers. In this study, we investigated the potential of three copies of C3d as a new molecular adjuvant to enhance the immunogenicity of hCG beta protein antigen. The antibody response to the hCG beta-C3d3 fusion protein immunization was compared with those resulting from immunization with the hCG beta alone and the hCG beta plus CFA/IFA either in BALB/c mice or in C(57)BL/6J mice. Our results showed that the fusion of C3d3 to hCG beta protein antigen resulted in a significant elevation of the serum anti-hCG beta antibody level in the two mouse strains and the antibodies were capable of effectively neutralizing the bioactivity of hCG. The immunization with C3d3 as a molecular adjuvant favored Th2 bias of immune response. The immunity-enhancing effect of the C3d3 was 10-fold (initial) and 20-32-fold (booster) greater than CFA/IFA. These findings indicated that fusion of C3d3 to hCG beta, as a means of harnessing the adjuvant potential of the innate immune system, may improve immunogenicity of the hCG beta contraceptive vaccine, which is useful to produce a cost-effective vaccine and for the less-responsive population.

Gene fusion of molecular adjuvant C3d to hCGbeta enhances the anti-hCGbeta antibody response in DNA immunization.

OBJECTIVE: To express the hCGbeta-C3d3 fusion protein in a CHO cell continual expression system to investigate further the adjuvant effects of C3d on contraceptive vaccination. METHOD: We constructed a plasmid pcDNA3-hCGbeta-C3d3 which contains three copies of murine C3d cDNA and the hCGbeta gene by cloning the chimerical hCGbeta-C3d3 cDNA into the eukaryotic vector pcDNA3 downstream of the CMV promoter. The plasmid was transfected into a COS-7 cell transient expression system and a CHO cell continual expression system. RIA was used to detect hCGbeta in the culture supernatant. Western blot and Raji cell immunohistochemical assays were performed to evaluate the expressed protein. Then, 6-8-week-old female BALB/c mice were inoculated intramuscularly with pcDNA3-hCGbeta and pcDNA3-hCGbeta-C3d3, and ELISA was used to assess anti-hCGbeta IgG antibody in serum. RESULTS: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGbeta-C3d3, 1.0x10(5) cells could secrete 152 ng of the recombinant protein (calculated by hCGbeta contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/10(6) cells/48 h. The hCGbeta-C3d3 protein was purified by anti-hCGbeta immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGbeta and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGbeta IgG antibody titer in the pcDNA3-hCGbeta-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGbeta immunized group. CONCLUSION: We have expressed the hCGbeta-C3d3 protein successfully, both in a transient expression system (COS-7 cells) and in a stable expression system (CHO cells). The C3d3 molecular adjuvant can enhance significantly the immunogenecity of hCGbeta antigen in DNA immunization.

Inoculation of Lactobacillus expressing hCG beta in the vagina induces an anti-hCG beta antibody response in murine vaginal mucosa.

OBJECTIVES: To test the possibility of vaccination with lactobacillus expressing hCG beta antigen administered by vaginal mucosal immunization. METHODS: A plasmid pIlac-hCG beta was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCG beta protein. RIA was used to detect hCG beta in the culture supernatant and cell lysate. Western blotting was performed to evaluate the expressed protein of interest. Female BALB/c mice aged 6-8 weeks received inoculations in the vagina of the recombinant L. casei CECT5276. ELISA was used to determine the anti-hCG beta IgA antibody in vaginal lavage fluid from the BALB/c mice after vaginal mucosal immunization. RESULTS: The pIlac alone appeared to have a higher efficiency than pIlac-hCG beta, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200 V and 1500 V. About 78.5% of the hCG beta protein was excreted into the culture supernatant. Excretion of hCG beta was most efficient when the pH of the culture medium was adjusted to around 7.0 and the concentration of lactose was around 1%. The hCG beta protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCG beta IgA antibody continued to be found in the vaginal lavage fluid for 2 weeks following a booster vaginal inoculation. The splenic lymphocytes of the mice immunized with hCG beta through the vagina underwent a proliferative reaction to hCG antigen restimulation in vitro. Interferon gamma (IFN-gamma) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG beta than after vaginal mucosal immunization of L. casei alone. CONCLUSIONS: Vaginal immunization of lactobacillus expressing hCG beta induced an anti-hCG beta antibody response in the murine vaginal mucosa. Induction of the antigen-specific antibodies in the reproductive tract following vaginal inoculation of recombinant lactobacillus will lead to the development of a safe, efficient, and easy-to-use form of immunocontraception.

[C3d molecular adjuvant increases the immunity of human chorionic gonadotropin beta DNA contraceptive vaccination and changes its immune response from Th1 to Th2].

OBJECTIVE: To testify the effect of C3d molecular adjuvant on the immunogenicity of human chorionic gonadotropin beta (hCG beta) DNA vaccination as well as the mode of immune response. METHODS: BALB/c mice aged 6 weeks were immunized intramuscularly two times at an interval of 3 weeks with by the plasmid pcDNA3 (A1-3 groups), pcDNA3-hCG beta (B-3 groups), pcDNA3-hCG beta-C3d3 (C1-3 groups), or pCMV4-hCG beta-C3d3 (D1-3 groups), at dosage of 5 pmol, 10 pmol, and 20 pmol, respectively. Three weeks after the second vaccination the animals were killed, specimens of their peripheral blood were extracted to determine the anti-hCG beta antibody titer by indirect ELISA. Their spleen cells were harvested and stimulated in vitro by hCG antigen for 24 hours. The Th1/Th2 cytokines in the culture supernatant were determined by ELISA. RESULTS: At the dosage of 20 pmol, C3d molecular adjuvant significantly enhanced the anti-hCG beta antibody titer. The utmost anti-hCG beta antibody titer of C3 group was 1:450, 9 times higher than that of B3 group, and the utmost anti-hCG beta antibody titer of D3 group was 1:12 150, 243 times higher than that of B3 group. Stimulated in vitro by 5,000 IU hCG beta antigen, the splenic cells of the C3 and D3 immunization group produced significantly lower IL-2, INF-gamma and TNF-alpha than those of the B3 immunization group (P < 0.01 or P < 0.05). The IL-4 level of the C3 group was higher than that of the B3 group while the IL-10 level of the D3 group was significantly higher than that of the B3 group (P < 0.01). CONCLUSIONS: The C3d molecular adjuvant increases significantly the hCG beta immunogenicity of hCG beta DNA vaccination; meanwhile decreases the secretion of Th1 cytokines (IL-2, INF-gamma, and TNF-alpha), and increases the expression of Th2 cytokines (IL-4 and IL-10) in response to hCG antigen. So C3d changes the anti-hCG immune response from Th1 type to Th2 type.

[The expression of chemokine receptors in CD56(bright) CD16- natural killer cells and the mechanism of their recruitment in decidua].

OBJECTIVE: To investigate the transcriptions of 18 chemokine receptors in human decidual natural killer (NK) cells and explore the possible mechanisms of preferential accumulation of CD56(bright)CD16(-)NK cells in decidua during first-trimester pregnancy. METHODS: Villi and decidual tissue were collected from normal pregnant women with 5 approximately 10 gestational weeks by artificial abortion. The decidual CD56(bright)CD16(-)NK cells were isolated by immune magnetic beads. The transcription levels of 18 chemokine receptors in the decidual CD56(bright)CD16(-)NK cells were assessed by reverse transcription-polymerase chain reaction (RT-PCR). After the high expression of CXCR4 and CXCR3 mRNA in these cells was found, the expression of stromal cell-derived factor-1 (SDF-1), the specific ligand of CXCR4, in first-trimester human placenta was detected by in situ hybridization and immunohistochemistry. The concentration of SDF-1alpha in the supernatant of culture of isolated trophoblast cells derived from the first-trimester human placentas was measured by ELISA. The chemotaxis of SDF-1 to decidual CD56(bright)CD16(-)NK cells was tested in Transwell, and the chemotactic activity was quantitatively examined. RESULTS: Among the 18 chemokine receptors, CXCR4 and CXCR3 were found highly transcribed in decidual CD56(bright)CD16(-)NK cells. The concentration of SDF-1alpha in the supernatant was 385 ng/L +/- 91 ng/L after trophoblast cells had been cultured for 60 hours. Both rhSDF-1alpha and supernatants in the culture of trophoblast cells exhibited chemotactic activity on decidual CD56(bright)CD16(-)NK cells. When the concentration of rhSDF-1alpha was 10 micro g/L the number of cells that entered the lower chamber of Transwell accounted for 22.9% +/- 4.3% of the total calls. CONCLUSION: First-trimester human trophoblast cells produce SDF-1, which in turn endows the trophoblast cells with the capacity to attract decidual CD56(bright)CD16(-)NK cells highly expressing CXCR4. This activity contributes to the recruitment of decidual lymphocytes and may be used at a local level to manipulate the microimmune environment at the materno-fetal interface.