Objective: To compare the effects of treatment of tubal pregnancy (TP)and its following second pregnancy by intratubal methotrexate injection (IMI) alone and combination of IMI with Chinese herbal medicine. Metlmds: Thirty-five patients sudferiug from unruptured TP were divided into two groups at random, to the 19 patients in the treated group, the treatment of combined IMI with Ectopic Pregnancy decoction No.2 (EP2, a traditional Chinese medical decoction) was applied, and to the other 16 patients in the control group, IMI alone was applied for control. Sexum concentrations of h-man chorionic gonadotro-pin (HCG), size of the gestational sac, existent time of fetal cardiac beat and peritoneal fluid were measured before and after treatment. And hysterosalpingography were performed 6 months after pnding the treatment to verify the presence of tubal obstruction and the condition of relapse. Results: The treat-ment of all the 35 women was suceessful. The recovery duration of serum β-HCG, disappeerance duration of TP sac and existent time of peritoneal fluid in the treated group were 20.04-7.8 days, 1.24-0.7 months and 10.74-2.9 days respectively, which were significantly different from those in the control group (24.4 4-8.1 days, 3.64-1.7 months and 19.14-3.2 days respectively(P＜0.05, P＜0.01 and P＜0.05 respec-β-tivdy), but the existent time of fetal cardiac beat in the two groups (8.84-1.9 days vs 9.0 4-1.3 days) was not significantly different (P~0.05). The post-treatment oviduct obstructive rate in the two groups was 10.5% and 43.8% respectively, that in the treatment group was less significant (P＜0.05). The relapse rate of EP in the treatment group was insignificantly different from that in the control group (5.3% vs 18.8%,P＜0.05).usion: The two therapies (IMI alone and IMI combined with EP2) could obtain e-qual efficacy in curing TP. Compared with IMI alone, the combined therapy appears to have the effects of accelerating the resorption of gestational sac and peritoneal fluid, improving the patency of fallopian tube and ameliorating the cirolm~tance of pregnancy, which is hvorable to improvement of the re-pregnancy rate and reduction of the re-occurrence of ectopic pregnancy as well as to the enhancement of the effect of IMI in killing trophocytes. But there is not enough proof to show the potency of EP2 in killing embryo.

Objectives: To test the possibility of vaccination with lactobacillus expressing hCGβ antigen administered by vaginal mucosal immunization. Methods: A plasmid pIlac-hCGβ was constructed and then transfected into Lactobacillus casei CECT5276, which stably expressed hCGβ protein. RIA was used to detect hCGβ 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-hCGβ 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-hCGβ, and the highest transfection efficiency of both plasmids was at pulse voltages of 1200V and 1500V. About 78.5% of the hCGβ protein was excreted into the culture supernatant. Excretion of hCGβ 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 protein in the vaginal lavage fluid of these BALB/c mice was positive on the third day after vaginal inoculation. Anti-hCGβ 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 hCGβthrough the vagina underwent a proliferative reaction to hCGβ antigen restimulation in vitro. Interferon gamma (IFN-γ) and interleukin (IL)-4 were secreted at higher levels after vaginal mucosal immunization of L. casei expressing hCG than after vaginal mucosal immunization of L. casei alone. Conclusions: Vaginal immunization of lactobacillus expressing hCG induced an antihCGβ 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.

More than 70% of decidual lymphocytes are NK cells characterized by CD56brightCD16- phenotype, but the mechanisms by which these NK cells are recruited in the decidua are still almost unrevealed. In this study, we first analyzed the transcription of 18 chemokine receptors in the first-trimester decidual CD56brightCD16- NK cells. Among these receptors, CXCR4 and CXCR3 were found highly transcribed, and the expression of CXCR4 was verified in most of the decidual CD56brightCD16-NK cells by flow cytometry. The first-trimester human trophoblasts were found expressing CXCL12/stromal cell-derived factor 1, the specific ligand of CXCR4, by way of in situ hybridization and immunohistochemistry. The primary cultured trophoblast cells were also found to secrete stromal cell-derived factor 1-spontaneously, and its concentration was 384.6-90.7pg/ml after the trophoblast cells had been cultured for 60 h. All of the ligands for CXCR3 were below the minimal detectable concentration when trophoblast cells were cultured for up to 48 h. Both recombinant human SDF-1-and supernatants of the cultured trophoblast cells exhibited chemotactic activity on decidual CD56brightCD16- NK cells. Our findings suggest that human first-trimester trophoblast cells produce CXCL12, which in turn chemoattracts decidual CD56brightCD16- NK cells. This activity could contribute to the recruitment mechanism of decidual lymphocytes, especially CD56brightCD16-NK cells, in decidua, and may be used at a local level to modulate the immune milieu at the materno-fetal interface.

Objective: To express the hCGβ-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-hCGβ-C3d3 which contains three copies of murine C3d cDNA and the hCGβ gene by cloning the chimerical hCGβ-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 hCGβ 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-hCGβ and pcDNA3-hCGβ-C3d3, and ELISA was used to assess anti-hCGβ IgG antibody in serum. Results: In 72 h after COS-7 cells were transfected with the plasmid pcDNA3-hCGβ-C3d3, 1.0/105 cells could secrete 152 ng of the recombinant protein (calculated by hCGβ contained). The transfected CHO cells, which were then screened by G418, could continuously secrete the fusion protein at 660 ng/106 cells/48 h. The hCGβ-C3d3 protein was purified by anti-hCGβ immunoaffinity chromatography. Raji cell immunohistochemical assay demonstrated that both the hCGβ and C3d3 were successfully fused. After DNA immunization intramuscularly, the anti-hCGβ IgG antibody titer in the pcDNA3-hCGβ-C3d3 immunized group was 243-fold higher than that of the pcDNA3-hCGβ immunized group. Conclusion: We have expressed the hCGβ-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 hCGβ antigen in DNA immunization.

Human chorionic gonadotropin (hCG) has been used as an anti-fertility vaccine and as a target for cancer immunotherapy. We have explored the use of three copies of C3d in DNA vaccine as molecular adjuvant to improve the immunogenicity of this hormone in previous work and found that the immune response induced by pcDNA3-hCGβ-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGβ following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGβ-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGβ and hCGβ-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGβ, pCMV4-hCGβ, pcDNA3-hCGβ-C3d3 and pCMV4-hCGβ-C3d3, respectively, at 25, 50 and 100 pmol dose, and further analyzed the levels of Th1 and Th2 cytokines produced by spleen lymphocytes of the immunized mice upon hCG restimulation in vitro. It was found that pCMV4 vector achieved 1.3-1.5-fold higher protein expression and raised 1.1-1.2 (primary) and 1.2-1.3 (booster) logs higher titer of anti-hCGβ IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGβ-C3d3-DNAs elicited the highest titer of hCGβ-specific antibody among the serial doses and the immune response induced by pCMV4-hCGβ-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGβ-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGβ at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGβ-C3d3-DNAs and the ratio of IL-4/IFN-γ showed a Th2 bias of immune response in the mice immunized with hCGβ-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGβ, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGβ DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3. Copyright Sons, Ltd.