Enlarged perivascular spaces, neuroinflammation and neurological dysfunction in NMOSD patients.

Background and objectives: Cerebrospinal fluid (CSF) and interstitial fluid exchange along a brain-wide network of perivascular spaces (PVS) termed the 'glymphatic system'. The aquaporin-4 (AQP4) water channels abundantly expressed on astrocytic endfeet play a key role in the CSF circulation in the glymphatic system. Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory demyelinating autoimmune disease of the central nervous system (CNS) featured with a specific autoantibody directed against AQP4 in most of patients. Anti-AQP4 antibodies are likely resulting in the impairment of the brain glymphatic system and the enlargement of PVS in NMOSD patients. In the current study, we aimed to demonstrate the features of EPVS detected by MRI and its association with the CSF anti-AQP4 antibody titer, CNS inflammatory markers, and disease severity in NMOSD patients. Methods: We conducted a retrospective review of a consecutive cohort of 110 patients with NMOSD who had brain MRI. We assessed the correlation of EPVS with markers of neuroinflammation, blood-brain barrier (BBB) function and severity of neurological dysfunction in patients. We used multivariate logistic regression analysis to determine the independent variables associated with disease severity. Results: The median number of total-EPVS was 15.5 (IQR, 11-24.2) in NMOSD patients. The number of total-EPVS was significantly related to EDSS score after correcting for the effects of age and hypertension (r=0.353, p<0.001). The number of total-EPVS was also significantly associated with the titer of CSF anti-AQP4 antibody, the albumin rate (CSF/serum ratios of albumin), the CSF albumin, IgG and IgA levels. Logistic regression analysis showed that total-EPVS and serum albumin level were two independent factors to predict disease severity in NMOSD patients (OR=1.053, p=0.028; OR=0.858, p=0.009 respectively). Furthermore, ROC analysis achieved AUC of 0.736 (0.640-0.831, p<0.001) for total-EPVS to determine severe NMOSD (EDSS 4.5-9.5). Discussion: In our cohort, we found a relationship between EPVS and neuroinflammation and BBB function in NMOSD. Moreover, EPVS might independently predict neurological dysfunction in patients with NMOSD.

Human Umbilical Cord Mesenchymal Stem Cells to Treat Neuromyelitis Optica Spectrum Disorder (hUC-MSC-NMOSD): A Study Protocol for a Prospective, Multicenter, Randomized, Placebo-Controlled Clinical Trial.

Background: Neuromyelitis Optica spectrum disorder (NMOSD) is severe relapsing and disabling autoimmune disease of the central nervous system. Its optimal first-line treatment to reduce relapse rate and ameliorate neurological disability remains unclear. We will conduct a prospective, multicenter, randomized, placebo-controlled clinical trial to study the safety and effectiveness of human umbilical cord mesenchymal stem cells (hUC-MSCs) in treating NMOSD. Methods: The trial is planned to recruit 430 AQP4-IgG seropositive NMOSD patients. It consists of three consecutive stages. The first stage will be carried out in the leading center only and aims to evaluate the safety of hUC-MSCs. Patients will be treated with three different doses of hUC-MSCs: 1, 2, or 5 × 106 MSC/kg·weight for the low-, medium-, and high-dose group, respectively. The second and third stages will be carried out in six centers. The second stage aims to find the optimal dosage. Patients will be 1:1:1:1 randomized into the low-, medium-, high-dose group and the controlled group. The third stage aims to evaluate the effectiveness. Patients will be 1:1 randomized into the optimal dose and the controlled group. The primary endpoint is the first recurrent time and secondary endpoints are the recurrent times, EDSS scores, MRI lesion numbers, OSIS scores, Hauser walking index, and SF-36 scores. Endpoint events and side effects will be evaluated every 3 months for 2 years. Discussion: Although hUC-MSC has shown promising treatment effects of NMOSD in preclinical studies, there is still a lack of well-designed clinical trials to evaluate the safety and effectiveness of hUC-MSC among NMOSD patients. As far as we know, this trial will be the first one to systematically demonstrate the clinical safety and efficacy of hUC-MSC in treating NMOSD and might be able to determine the optimal dose of hUC-MSC for NMOSD patients. Trial registration: The study was registered with the Chinese Clinical Trial Registry (CHICTR.org.cn) on 2 March 2016 (registration No. ChiCTR-INR-16008037), and the revised trial protocol (Protocol version 1.2.1) was released on 16 March 2020.

Single-Cell Characterization of Malignant Phenotypes and Developmental Trajectories of Adrenal Neuroblastoma.

Neuroblastoma (NB), which is a subtype of neural-crest-derived malignancy, is the most common extracranial solid tumor occurring in childhood. Despite extensive research, the underlying developmental origin of NB remains unclear. Using single-cell RNA sequencing, we generate transcriptomes of adrenal NB from 160,910 cells of 16 patients and transcriptomes of putative developmental cells of origin of NB from 12,103 cells of early human embryos and fetal adrenal glands at relatively late development stages. We find that most adrenal NB tumor cells transcriptionally mirror noradrenergic chromaffin cells. Malignant states also recapitulate the proliferation/differentiation status of chromaffin cells in the process of normal development. Our findings provide insight into developmental trajectories and cellular states underlying human initiation and progression of NB.

Serum albumin level is associated with the severity of neurological dysfunction of NMOSD patients.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory autoimmune disease of the central nervous system. Serum albumin (SA) has antioxidant, immunomodulatory and anti-inflammatory effects. However, the roles of SA in NMOSD have not been studied. The current study aimed to clarify the association of SA with disease severity and prognosis in NMOSD patients. METHODS: Serum levels of albumin were measured by Bromcresol Green method. Serum level measurements of interleukins were performed using enzyme-linked immunoassay (ELISA) method. RESULTS: Of all the 130 NMOSD patients, 96 patients were in the acute phase while 34 patients were in the remission phase of disease at the time of sampling. SA concentration was significantly correlated with EDSS score in patients in the acute phase but not in remission phase (r = - 0.388, p < 0.001 and r = - 0.467, p = 0.809, respectively). Logistic analysis revealed that SA was the only significant factor to predict severe NMOSD (EDSS 8.0-9.5) OR = 0.698, 95%CI 0.563-0.865, p = 0.001) after adjustment of other confounding factors. Furthermore, SA was negatively correlated with the serum level of IL-33 (r = -0.438, p = 0.016) in the acute phase of NMOSD patients. CONCLUSION: The current study found that low level of SA was an independent indicator of more severe neurological deficit in patients in acute phase of NMOSD. SA concentration was negatively correlated with the serum level of IL-33 in the acute phase of the disease, which implies that SA might participate in the immunopathology of NMOSD partly through its interaction with IL-33.

Th2 axis-related cytokines in patients with neuromyelitis optica spectrum disorders.

AIMS: Neuromyelitis optica spectrum disorder (NMOSD) is an inflammatory autoimmune disease of the central nervous system. Increasing evidence indicates that NMOSD is a Th2- and Th17-dominant disease. IL-25, IL-31, and IL-33 are three newly found Th2-related cytokines, and their roles in the pathogenesis of NMOSD have not been studied. This study aimed to measure the serum levels of IL-25, IL-31, and IL-33 in patients with NMOSD and evaluate their clinical implications. METHODS: Serum was collected from patients with NMOSD (n = 48) and healthy controls (HC, n = 28). Serum level measurements of IL-25, IL-31, IL-33, IL-17A, and IL-6 were performed using enzyme-linked immunoassay (ELISA) method. RESULTS: The serum levels of IL-25, IL-31, and IL-33 were significantly higher in patients with NMOSD as compared to HC. The serum level of IL-31 was significantly correlated with IL-17A (r = 0.382,P = 0.009) in patients with NMOSD; the latter is a critical cytokine in the pathogenesis of NMOSD. The serum level of IL-33 was higher in patients with characteristic brain lesions than patients without (307 pg/mL vs 166 pg/mL, P = 0.028). Furthermore, the serum level of IL-33 in the acute phase of the disease was positively correlated with annualized relapse rate (r = 0.364, P = 0.04). CONCLUSION: We found higher serum levels of IL-25, IL-31, and IL-33 in patient with NMOSD as compared to healthy controls. The serum level of IL-33 during acute phase was associated with more past attacks in patients with NMOSD.

Mouse endometrial stromal cells and progesterone inhibit the activation and regulate the differentiation and antibody secretion of mouse B cells.

The regulation mechanism for the B cells in the female reproductive tract (FRT) is unclear now. The aim of this study was to analysis the effect of progesterone and mouse endometrium stromal cells (ESCs) on B cells and explore it roles in modulating B cells-mediated immune responses. We primary isolated mouse ESCs from endometrium of BALB/c mice and B cells from spleen cells of BALB/c mice, and then constructed these two kind of cells co-culture system, and treated with or without progesterone. We found that both treatment with progesterone and co-culture with ESCs reduced the expression of co-stimulatory molecules CD80 and CD86 on mouse B cells from spleen cells. In addition, the expression of CD138 (syndecan-1) on B cells was increased after co-culture with ESCs, however, progesterone could partly reduce this effect. Unlike progesterone, ESCs alone promoted the proliferation and stimulated the secretion level of antibodies IgG and IgA of B cells. Our current results progesterone and ESCs could inhibit the activation of B cells through deceasing CD80 and CD86 expression, regulated the differentiation status of B cells by up-regulating the expression of CD138 together, and might further inhibit the antigen presentation function of B cells, which is beneficial to the establishment of fertilization and pregnancy. In addition, ESCs also promoted the proliferation and antibody secretion, which might participate in the resisting infections during non pregnancy and pregnancy.

[Clinical features and prognostic factors in patients with leptomeningeal metastases].

OBJECTIVE: To investigate the clinical characteristics and prognostic factors of leptomeningeal metastases (LM) from solid tumors and to develop better treatment strategies. METHODS: The clinical characteristics and follow-up results of 77 cases of leptomeningeal metastases (LM) from solid tumors diagnosed and treated in our hospital from 2002 to 2011 were retrospectively analyzed. Clinical characteristics, treatment methods and overall survival were analyzed using Kaplan-Meier method and Cox regression model. RESULTS: The median survival time for all the patients was 88 days. KPS score, control of the primary tumor and systemic treatment were correlated with survival time for the patients (P < 0.05 for all). The median survival time of systemic treatment was 150 d and those without systemic treatment (chemotherapy and/or targeted therapy) after LM was 60 d (P = 0.001). Systemic therapy combined with local treatment (radiotherapy to the meninges or intrathecal chemotherapy) further improved the survival time of patients. Multivariate analysis showed that KPS and short-term therapeutic response for the LM were independent prognostic factors (P < 0.05 for both). CONCLUSIONS: KPS and short-term therapeutic response are independent prognostic factores for leptomeningeal metastases from solid tumors. Systemic chemotherapy or targeted therapy can prolong the survival time. Systemic treatment (chemotherapy and/or targeted therapy) combined with radiation therapy or intrathecal injection may further improve the clinical outcomes.

[An analysis of risk factors for ischemic stroke of different age and gender].

OBJECTIVE: To compare distribution difference in risk factors of patients with first-ever ischemic stroke (IS) of different age and gender. METHODS: A total of 1027 patients admitted to the neurological department in Shanghai Renji Hospital with first-ever IS were recruited and divided into young adult group (< 50 years old), middle-aged group (50 - 80 years old), and very old group (> 80 years old) according to their ages. Risk factor analysis included history of smoking, high alcohol consumption, hypertension (HT), diabetes mellitus (DM), heart diseases, atrial fibrillation (AF) and family history of cardiovascular diseases. RESULTS: Female patients were globally older than male patients (71.1 vs 65.7, P < 0.001) at the first attack of IS and having higher prevalence of DM (26.8% vs 19.2%, P = 0.004), heart diseases (28.8% vs 19.2%, P < 0.001) and AF (7.6% vs 3.9%, P = 0.009). However, female patients were less likely to drink heavily (1.0% vs 31.6%, P < 0.001) or smoke (4.4% vs 59.9%, P < 0.001) than the male patients. The rates of smoking and heavy drinking in young adult group were higher than that in other two groups. Patients in very old group had higher prevalence of heart diseases and AF but lower proportion of positive family cardiovascular diseases history than patients in other two groups. HT and DM were equally frequent among three groups. In young adult group, female patients were more likely to have heart diseases and family history of heart diseases (P = 0.015 and P = 0.048). In middle-old group, HT, DM, heart disease and AF were more common in women than in men (P = 0.021, P = 0.004, P = 0.001 and P = 0.039). CONCLUSION: There are differences in risk factor distribution in patients with first-ever IS of different age and gender. Therefore, screening and health education should be performed in allusion to different risk factors.

Age- and gender-specific prevalence of risk factors in patients with first-ever ischemic stroke in china.

Background. Evidences are accumulating that age and gender have great impact on the distribution of stroke risk factors. Such data are lacking in Chinese population. Methods. 1027 patients with first-ever ischemic stroke (IS) were recruited and divided into young adult (<50 years), middle-aged (50∼80 years), and very old (>80 years) groups according to stroke onset ages. Vascular risk factors were collected and compared among groups. Results. Female patients were globally older than male patients at stroke onset and having higher prevalence of diabetes mellitus (DM), heart diseases, and atrial fibrillation (AF). However, females were less likely to drink heavily or smoke than males. Young patients had a much higher proportion of smoking and drinking than middle-aged and very old patients and the highest family history of hypertension, while very old patients had the highest prevalence of heart diseases and AF but lowest proportion of positive family history of vascular diseases. Hypertension and DM were equally frequent among three groups. Conclusion. Our study showed that vascular risk factors had a specific age and gender distribution pattern in Chinese IS patients. Secondary prevention strategy should emphasize on the control of different risk factors based on patient's age and gender.

Downregulation of miR-199a may play a role in 3-nitropropionic acid induced ischemic tolerance in rat brain.

MicroRNAs (miR) are single-stranded short RNA molecules that regulate gene expression by degradation or translational repression of mRNA. It has been reported that the downregulation of miR-199a plays an important role in cardiac ischemic tolerance. We examined the expression of miR-199a after 3-nitropropionic acid (3-NPA) preconditioning in rat brain. 3-NPA (20mg/kg), an irreversible inhibitor of succinate dehydrogenase, was injected intraperitoneally to induce ischemic tolerance in rats. For comparison, the control group received intraperitoneal injections of vehicle (0.9% sodium chloride). Quantitative RT-PCR assay was applied to detect the expression of miR-199a. Luciferase reporter assays and Western blotting were used to verify the target genes of miR-199a. In cortex and striatum, miR-199a was downregulated at two separate time intervals (the 2nd and 4th day), while in the hippocampus, it was downregulated on the 2nd day after 3-NPA preconditioning. The maximum reduction of miR-199a was 66.3% in striatum (4th day), 54.9% in hippocampus (2nd day), and 27.6% in cortex (2nd day). The level of sirt1 protein, a putative target of miR-199a and a known mediator of neuroprotective effect in brain ischemic tolerance, decreased significantly in hippocampal neurons by overexpression of miR-199a, while it increased with knockdown of miR-199a. Taking these results together, we hypothesize miR-199a may have a role in the formation of cerebral ischemic tolerance.

Fusion of hC3d3 to hCGbeta enhances responsiveness in vitro of human peripheral immunocompetent cells upon the antigen primary challenge.

Contraceptive vaccines based on hCGbeta have not met clinical application because of poor immunogenicity. In the present study, the eukaryotic expression vectors pCI-gs-signal-6His-hCGbeta and pCI-gs-signal-6His-hCGbeta-hC3d3 were constructed, and transfected into CHO cells with aid of Lipofectaine 2000 reagent to gain the secretory recombinant protein. Isolated B cells from human peripheral blood, combined B cells with T cells, and PBMC were treated in vitro, respectively, with 1 nM, 10 nM, 100 nM hCGbeta, hCGbeta-hC3d3 or PWM for 12 days. Immunoglobulin (Ig) and anti-hCG antibody levels in the supernatant were measured by an indirect enzyme-linked immunosorbent assay (ELISA). The expressions of CD80/CD86 on B cells, and CD154/CD25 on T cells, were analyzed by flow cytometry (FCM), and IL-2 production was assayed by ELISA. It was found that the Ig levels in the B-cell supernatants, the combined B with T cells, and PBMC treated with 100 nM hCGbeta-C3d3 fusion protein were 4-fold, 10-fold and 10.9-fold more, respectively, than that of hCGbeta. The anti-hCG antibody could be produced in the combined B cells with T cells, as well as PBMC challenged with 100 nM hCGbeta-C3d3, but no anti-hCG antibody was produced in the challenge with hCGbeta. The hCGbeta-hC3d3 fusion protein enhanced the expression of CD80 and CD86 on B cells, especially CD86 (P<0.05), and significantly increased the expression of CD154 and CD25 molecules on T cells compared to that of hCGbeta (P<0.05). The hCGbeta-hC3d3 promoted human PBMC producing more IL-2 than hCGbeta. These findings indicate that the fusion of hC3d3 to hCGbeta, as a means of harnessing the adjuvant potential of the innate immune system, may contribute to a more efficient humoral immune response, and might provide a potential application of protein vaccine strategies in humans in the future.

[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.

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.

[A randomized study comparing the efficacy and bleeding pattern of Implanon and Norplant hormonal contraceptive implant].

OBJECTIVE: To compare the contraceptive efficacy, tolerability and bleeding patterns of Implanon and Norplant. METHODS: One hundred healthy female volunteers received either Implanon (75 cases) or Norplant (25 cases) for 4 years, followed up with complain, bleeding patterns, hemoglobin, blood pressure, weight. Record the time of insert and remove. RESULTS: There were no pregnancies during the study. Per 90-day reference period, the average number of bleeding-spotting days with Implanon decreased from 33 day in the first period to 21 day in the last year, and Norplant decreased from 31 day to 20 day. The average bleeding number of times was 2.25 of Implanon and 2.99 of Norplant per reference period (P < 0.05). The mean insert time was 11 second of Implanon and 103 second of Norplant. The mean remove time was 27 second of Implanon and 102 second of Norplant (P < 0.001). CONCLUSION: Both contraceptive systems demonstrated excellent contraceptive efficacy and were well tolerated. Because of its single-rod design, Implanon was significantly quicker to insert and remove than the multiple-capsule system.