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.

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.

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.

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.

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.

[Gene conjugation of molecular adjuvant C3d3 to hCGbeta enhanced the anti-hCGbeta humoral immune response via upregulation of CXCR4 expression on splenic B cells in DNA vaccination].

To investigate mechanism of the anti-hCGbeta humoral immune responsive enhancement by gene conjugation of the molecular adjuvant C3d3 to hCGbeta in DNA immunization, BALB/c mice were inoculated intramuscularly with pCMV4-hCGbeta-C3d3, pCMV4-hCGbeta and pCMV4, respectively. The titers of anti-hCGbeta IgG/IgA antibody in serum were determined by indirect ELISA. The IgG/IgA ASCs levels were evaluated by ELISPOT. The expressions of chemokine receptors on B cells were analyzed by RT-PCR, and CXCR4 expression was analyzed respectively by RT-PCR and FCM. The expression of CXCL12 in spleen was investigated respectively by RT-PCR and ELISA. We found that anti-hCGbeta IgG antibody titer in serum after pCMV4-hCGbeta-C3d3 immunization was significantly higher than that of pCMV4-hCGbeta immunization. But the anti-hCGbeta IgA antibody titers appeared no difference between the two immunized groups. The level of IgG ASCs in spleen of pCMV4-hCGbeta-C3d3 immunization was significantly higher than that of pCMV4-hCGbeta immunization,but the levels of IgA ASCs appeared no difference between the two groups. The expression of CXCR4 on B cell increased after pCMV4-hCGbeta-C3d3 immunization, and significantly higher than that of pCMV4-hCGbeta immunization. The rate of CXCR4+ cell was correlated to the numbers of the ASC (r = 0.966, P < 0.05). CXCL12 expression increased after pCMV4-hCGbeta and pCMV4-hCGbeta-C3d3 immunization, and appeared no difference between the two groups. These results above indicate that gene fusion of the molecular adjuvant C3d3 to hCGbeta can significantly enhance the antigen-specific antibody response, and the level of IgG ASCs in spleen via upregulation of CXCR4 expression on splenic B cells in DNA vaccination.

Molecular adjuvant C3d3 improved the anti-hCGbeta humoral immune response in vaginal inoculation with live recombinant Lactobacillus expressing hCGbeta-C3d3 fusion protein.

To enhance the contraceptive efficiency of human chorionic gonadotrophin (hCG)-beta contraceptive vaccine, we coupled hCG-beta gene with molecular adjuvant C3d3, and cloned into live Lactobacilli (Lb.) to express fusion protein hCGbeta-C3d3. The recombinant Lb. could survive in BALB/c murine vagina for at least 3 weeks. After inoculating BALB/c and C57BL/6 mice via vagina, we found that the antibody titer peaks induced by the Lb.hCGbeta-C3d3 inoculation were higher significantly than the Lb.hCGbeta. T and B cells in spleen and vagina were significantly increased, and anti-hCGbeta IgG and IgA antibody-secreting cells in uterus and vagina were significantly increased compared to the control in different strain mice. Our study shows that the C3d3 can display apparent adjuvant efficiency to induce more powerful humoral response to the hCGbeta antigen in vaginal mucosal immunization.

[Molecular adjuvant C3d up-regulates both B7-1 and B7-2 expression on Raji cells].

To explore modulation of the molecular adjuvant C3d in the hCGbeta -C3d3 fusion protein in costimulatory molecule expression on Raji cells by linking to the surface molecule CD21. Raji cells, B cell line, were incubated with the purified hCGbeta-C3d3, hCGbeta or C3d3 protein for 15 hours respectively in the interference of anti-CD21 monoclonal antibody or not. The culture concentration of each group was 10, 30, or 90 microg/ml respectively. The expression of both B7-1 and B7-2 on Raji cells were analyzed by flow cytometric assay. It was observed that compared to hCGbeta or C3d3 protein, hCGbeta-C3d3 up-regulated both B7-1 and B7-2 expression on the Raji cells, and the effect was in a dose-dependent manner. The up-regulation effect was blocked efficiently by anti-CD21 mAb. The results showed that molecular adjuvant C3d may up-regulate both B7-1 and B7-2 expression on Raji cell via C3d/CD21/CD19 complex, thus enhance the antigen presentation of B cell, and the interaction between B cell and T cell.

Gene conjugation of molecular adjuvant C3d3 to hCGbeta increased the anti-hCGbeta Th2 and humoral immune response 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-hCGbeta-C3d3 has been enhanced 243-fold compared with pcDNA3-hCGbeta following DNA immunization in BALB/c mice. In the present study, a new functionally active DNA vaccine of hCGbeta-C3d3 chimera based on pCMV4 vector has been described. We compared the expression efficiency of pCMV4 and pcDNA3 eukaryotic vectors for hCGbeta and hCGbeta-C3d3 fusion protein and the immune response of mice immunized with pcDNA3-hCGbeta, pCMV4-hCGbeta, pcDNA3-hCGbeta-C3d3 and pCMV4-hCGbeta-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-hCGbeta IgG than pcDNA3. Mice vaccinated with 50 pmol of hCGbeta-C3d3-DNAs elicited the highest titer of hCGbeta-specific antibody among the serial doses and the immune response induced by pCMV4-hCGbeta-C3d3 were, respectively, 1.3, 1.3 and 1.2 logs higher than that of pcDNA3-hCGbeta-C3d3 and 2.2, 2.9 and 2.4 logs higher than that of pCMV4-hCGbeta at week 2 following the booster immunization. Moreover, we observed that the production of IL-4 and IL-10 increased in mice vaccinated with hCGbeta-C3d3-DNAs and the ratio of IL-4/IFN-(gamma) showed a Th2 bias of immune response in the mice immunized with hCGbeta-C3d3-DNAs. These findings indicated that gene fusion of C3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may improve the antigen-specific Th2 humoral immune response of the hCGbeta DNA vaccine and the pCMV4 vector is a more ideal eukaryotic vector for DNA vaccine than pcDNA3.

Mucosal inoculation of Lactobacillus expressing hCGbeta induces an anti-hCGbeta antibody response in mice of different strains.

To show that an anti-human chorionic gonadotrophin-beta (hCGbeta) antibody response can be induced by inoculating Lb. expressing hCGbeta through different mucosal pathways in mice of two strains, female BALB/c and C57BL/6 mice were immunized via vaginal, oral or nasal routes with 10(8), 10(9), and 10(10)Lb.hCGbeta (a recombinant Lactobacillus expressing hCGbeta). The mice were immunized twice with a booster in study week 3. An indirect ELISA was used to determine anti-hCGbeta IgG and IgA antibodies in vaginal lavage and serum, obtained from the 2nd to 8th week after the primary immunization. Flow cytometry was used to analyze the lymphocyte proliferation from these tissues, 1 week after the primary immunization. The hCGbeta antigen-specific antibody-secreting cells of spleen, uterus, and vagina were evaluated by enzyme-linked immunospot assay (ELISpot), 2 weeks after the booster. The analysis showed that 10(9) and 10(10)Lb.hCGbeta inoculations induced similar anti-hCGbeta antibody responses, while the three mucosal pathways induced similar antibody responses. The antiserum obtained after boosters with 10(9) and 10(10)Lb. hCGbeta was able to neutralize more than 100 ng/ml hCG antigen, both in BALB/c and C57BL/6 mice. The highest antibody titer induced by vaginal mucosal immunization was stronger than that obtained via the other mucosal pathways. The B cells in the vagina appeared to proliferate after vaginal immunization (P<0.05). The numbers of anti-hCGbeta IgG and IgA antibody-secreting cells in the uterus and vagina were greater than in the spleen. Therefore, the vaginal mucosal route appears to be a better immunization pathway to induce higher anti-hCGbeta antibody levels in the reproductive tract.

[The mechanism of the recipient maternal-fetal immuno-tolerance induced by the transferred paternal antigen-tolerant T cells].

To study the effect of adoptive transfer of paternal antigen-tolerant T cells on recipient reactive T cells, CBA/JxDBA/2 mating was recruited as an abortion-prone model, and CBA/JxBALB/c mating as a successful pregnancy model. The abortion-prone CBA/J females mated with DBA/2 males were injected intraperitoneally with rat anti-mouse CD80 and CD86 mAb or rat isotype IgG at day 4 after gestation (time of implantation). The purified T cells were obtained from spleen of the pregnant CBA/J mice using magnetic beads at day 9 after gestation and labeled with CFSE in vitro. The CFSE-labeled T cells were intravenously injected into other CBA/J females mated with DBA/2 males at day 4 after gestation. The proliferation of recipient splenocytes in response to DBA/2 stimulator cells was evaluated at day 9 after gestation in vitro, and the expressions of intracellular cytokines and costimulatory molecules in CFSE +/- T cells were analyzed by flow cytometry. The results showed that adoptive transfer of either paternal antigen-tolerant T cells or T cells from BALB/c-mated CBA/J mice significantly suppressed the proliferation of recipient splenocytes in response to DBA/2 stimulator cells and resulted in lower frequency of cells positive for IL-2, IFN-gamma, CD28 and higher frequency of IL-10,CTLA-4-producing cells in both CFSE+ CD3+ population and CFSE- CD3+ population compared with adoptive transfer of T cells from isotype IgG-treated CBA/J mice, whereas the frequency of IL-4-producing cells did not appear significant change. Our findings suggest that paternal antigen-tolerant T cells transferred in recipient not only function as antigen-specific suppresser cells but also disable the recipient reactive T cells, which co-suppresses maternal rejection to the allogeneic fetus, thus resulting in the decrease of the embryo resorption rate of the abortion-prone mice to that of the normal pregnancy mice.

[Cyclosporin a induces titin expression in human trophoblast cells through the MEK/ERK1/2 signal pathway].

To investigate the role of MEK/ERK1/2 signal pathway in the regulation of cyclosporin A(CsA) -induced titin expression in human trophoblast cells. With RT-PCR and Western Blot, We examined the titin expression level of human trophoblast cells treated with different concentrations of CsA for various duration, then detected total ERK1/2 and phosphorated ERK1/2 level with Western Blot, and observed effect of U0126 on transcription of titin mRNA in human trophoblast cells stimulated by cyclosporin A. It was found that CsA could activate ERK1/2 in time-dependent and dosage-dependent manner, and induced titin to be expressed in human trophoblast cells. U0126, a MEK inhibitor, inhibited the transcription of titin in dosage-dependent manner. These results indicated that MEK/ERK1/2 signal pathway may play an important role in the expression of titin in human trophoblast induced by cyclosporin A.

Human first-trimester trophoblast cells recruit CD56brightCD16- NK cells into decidua by way of expressing and secreting of CXCL12/stromal cell-derived factor 1.

More than 70% of decidual lymphocytes are NK cells characterized by CD56(bright)CD16(-) 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 CD56(bright)CD16(-) NK cells. Among these receptors, CXCR4 and CXCR3 were found highly transcribed, and the expression of CXCR4 was verified in most of the decidual CD56(bright)CD16(-) 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 1alpha spontaneously, and its concentration was 384.6 +/- 90.7 pg/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-1alpha and supernatants of the cultured trophoblast cells exhibited chemotactic activity on decidual CD56(bright)CD16(-) NK cells. Our findings suggest that human first-trimester trophoblast cells produce CXCL12, which in turn chemoattracts decidual CD56(bright)CD16(-) NK cells. This activity could contribute to the recruitment mechanism of decidual lymphocytes, especially CD56(bright)CD16(-) NK cells, in decidua, and may be used at a local level to modulate the immune milieu at the materno-fetal interface.

Blockade of CD86 signaling facilitates a Th2 bias at the maternal-fetal interface and expands peripheral CD4+CD25+ regulatory T cells to rescue abortion-prone fetuses.

Intervention in B7 (CD80/CD86)/B7-ligand (CD28/CTLA-4) pathways is an effective way of preventing unwanted immune responses, such as allograft rejection. Pregnancy maintenance represents maternal tolerance to the fetal allograft, which is accompanied by a type 2 helper cell (Th2) bias at the maternal-fetal interface. Here, the costimulatory signal of CD86 was selectively blocked, and that of CD80 was kept unimpaired by administration of anti-murine CD86 monoclonal antibody at the early gestational stage in abortion-prone CBA/JxDBA/2 matings and normal pregnant CBA/JxBALB/c matings. It was demonstrated that in vivo blockade of CD86 costimulation could suppress maternal immune attack to the fetus by shifting cytokines from Th1 predominance to Th2 bias at the maternal-fetal interface, and expanding peripheral CD4+CD25+ regulatory T cells, which play an important role in the development and maintenance of maternal-fetal tolerance. Furthermore, the expression of CD28 and its ligands CD80/CD86 on peripheral lymphocytes was down-regulated, whereas that of CTLA-4 was up-regulated, which might facilitate the suppressive effect of CD4+CD25+ regulatory T cells on the alloreactive T cells. The maternal-fetal immunotolerance induced by CD86 blockade decreased fetal resorption in CBA/JxDBA/2 matings, but did not affect normal pregnant CBA/JxBALB/c matings. These results suggest that selective blockade of CD86 costimulation leads to maternal immune tolerance to embryo antigen, and might contribute to a rational immunoregulatory regimen for recurrent spontaneous abortion.

[Enhancement of immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3].

To enhance the immunogenicity of hCGbeta protein vaccine and the hCG neutralization capacity of anti-serum by using the molecular adjuvant C3d3, the secreted 6his-hCGbeta-C3d3 fusion protein and 6his-hCGbeta were expressed in CHO cells and purified with Ni(2+)-chelating chromatography and Sephadex G-150 column. Then we investigated the potential of three copies of C3d as the molecular adjuvant of hCGbeta protein antigen. The antibody response to hCGbeta-C3d3 conjugates was compared with those resulting from immunization with hCGbeta alone and hCGbeta plus CFA/IFA in BALB/c mice. Our results showed that the antibody titer of hCGbeta-C3d3 was 1995-fold higher than that of hCGbeta alone and the anti-serum was capable effectively neutralizing the bioactivity of hCG. The immunity-enhancing action of C3d3 was 10-fold (primer) and 32-fold (booster) greater than CFA/IFA. These results indicated that C3d3 conjugates might be a better way to overcome the poor immunogenicity of hCGbeta contraceptive vaccine.

Adoptive transfer of paternal antigen-hyporesponsive T cells induces maternal tolerance to the allogeneic fetus in abortion-prone matings.

The embryo expresses paternal Ags foreign to the mother and therefore has been viewed as an allograft. It has been shown that anergic T cells generated by blocking of the CD28/B7 costimulatory pathway with anti B7-1 and anti B7-2 mAbs can be transferred as suppresser cells to prevent allograft rejection. Little is known, however, about the in vivo function of anti-B7-treated T cells after their transfer into abortion-prone mice in the maintenance of materno-fetal tolerance. In the present study, abortion-prone CBA/J females mated with DBA/2 males were administered anti-B7-1 and anti-B7-2 mAbs on day 4 of gestation (murine implantation window). The anti-B7-treated T cells subsequently were adoptively transferred into abortion-prone CBA/J mice. We demonstrated that costimulation blockade with anti-B7 mAbs at the time of implantation resulted in altered allogeneic T cell response and overcame increased maternal rejection to the fetus in the CBA/JxDBA/2 system. The transferred anti-B7-treated T cells appeared to be regulatory, decreasing responsiveness and generating clonal deviation in maternal recipient T cells. The transferred CFSE-labeled T cells were found to reside in the spleen and uterine draining lymph nodes, and a few were localized to the materno-fetal interface of the maternal recipient. Our findings suggest that the anti-B7-treated T cells not only function as potent suppresser cells, but also exert an immunoregulatory effect on the maternal recipient T cells, which cosuppresses maternal rejection to the fetus. This procedure might be considered potentially useful for fetal survival when used as an immunotherapy for human recurrent spontaneous abortion.

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.

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.

[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.

[Inducing peripheral materno-fetal immuno-tolerance by blockade of costimulatory signal at early stage of gestation of the abortion-prone murine model].

OBJECTIVE: To study effect of blockade of costimulatory signal CD(80), CD(86) at the early stage of gestation on the maternal peripheral immuno-tolerance status to paternal antigen and pregnant outcome of murine abortion-prone model. METHODS: The experiments were performed in two groups, using CBA/J x BALB/c matings as the normal pregnancy model and CBA/J x DBA/2 matings as the abortion-prone model. The pregnant CBA/J mice which had mated with DBA/2 or BALB/c male mice respectively were injected intraperitoneally with purified rat isotype IgG or purified rat anti-mouse CD(80), CD(86) mAb at day 4 of gestation (time of implantation). The proliferation response of maternal splenic immuno-competent cells to paternal splenic cells as stimulator was analyzed by one way mixed lymphocyte reaction, and IL-2 production was assayed by ELISA to evaluate the immuno-tolerance of maternal peripheral immuno-competent cells to paternal antigen at day 9 of gestation. The embryo resorption rate was counted at day 14 of gestation. RESULTS: In the CBA/J x DBA/2 matings, anti-CD(80), CD(86) mAb administered at day 4 of gestation reduced significantly the embryo resorption rate, and induced a significantly lower proliferation response and IL-2 production by maternal splenic immuno-competent cells to paternal antigen compared with rat isotype IgG. CONCLUSIONS: In vivo blockade of costimulatory signal at day 4 of gestation can induce the immuno-tolerance of maternal peripheral immuno-competent cells to paternal antigen, thus leading the embryo resorption rate of the abortion-prone model to that of normal pregnancy model.