Qualitative differences in sperm antibody responses in mice of different inbred strains and sexes

To better understand the immunogenetic basis and potential pathological consequences of anti-sperm humoral immunity, age-matched female mice of 9 different inbred strains were immunized with syngeneic sperm and were tested for qualitative (specificity) and quantitative (titer) antibody differences by radioimmunoassay, immunofluorescence and immunoblot techniques. All mice developed antisperm antibodies, although titers varied considerably between inbred strains. Antisperm antibodies produced in this study did not cross-react with membrane antigens on thymocytes, brain or immature testicular germ cells. Immunoblot tests identified 17 major sperm antigen bands; this approach also revealed considerable inter- and intra-strain variation in antisperm antibody specificities among female mice. In a parallel study C57BL/6 male mice demonstrated significantly lower antisperm antibody titers and an absence of response to certain sperm antigens in immunoblot tests when compared to age-matched females of the same inbred strain. These findings provide evidence that genetic factors (including sex) interact with environmental (nongenetic) factors in the control of immune responses to sperm antigens.     

Differences in immunogenicity indicating polymorphism of sperm antigens from mice of different inbred strains

Polymorphism of mouse sperm was investigated by analysis of immune sera generated in BALB/c female mice against sperm from 6 inbred strains. The immune sera were analyzed by immunofluorescence and Western blot techniques against sperm antigens from the 6 immunizing strains. Immunofluorescence revealed no differences in reactivity patterns or titers. However, several different reaction patterns were detected by Western blot technique which indicated that both the sperm extracts and the antisperm immune sera contained different components. Syngeneic (anti-BALB/c sperm) antisera showed far fewer reactive antibody species than allogeneic immune sera. The anti-BALB/c sera recognized an antigen of 23 kDa in sperm extracts from DBA/2J and C57BL/6 mice, and failed to react with an antigen of the same molecular weight when applied to sperm from A/J and 129/J mice, indicating antigenic differences between sperm from these inbred strains. Anti-C57BL/6 sera contained a unique antibody which reacted with an antigen of 80 kDa in all of the 6 sperm extracts, whereas others antisera did not detect this antigen. These findings indicate antigenic and immunogenic polymorphism in sperm from different inbred strains of mice.     

The anti-tumor immune responses induced by a fusion protein of ovarian carcinoma anti-idiotypic antibody 6B11ScFv and murine GM-CSF in BALB/c mice

Ovarian carcinoma anti-idiotypic antibody 6B11 was murine derived; we previously have cloned 6B11 single-chain Fv antibody (6B11ScFv) and constructed the 6B11ScFv/human granulocyte-macrophage colony stimulating factor (GM-CSF) fusion protein (designated as 6B11GM) to enhance the immunogenecity of the single-chain Ab(2). Because of the difference in species specificity between human GM-CSF and murine GM-CSF, there is no immune competent animal model on which the effect and metabolism of 6B11GM as a vaccine could be observed. In this study, 6B11mGM fusion gene was constructed by the fusing murine GM-CSF cDNA gene with 6B11ScFv. The fusion gene was cloned and expressed. The product of this gene is a fusion protein. It could specifically interact with the primary anti-ovarian carcinoma monoclonal antibody (COC166-9) and rat anti-mouse GM-CSF monoclonal antibody, respectively, and stimulate the growth of NFS-60 cells (a murine GM-CSF-dependent cell line). The specific anti-tumor immune response could be induced in BALB/c mice after immunized with anti-idiotypic fusion protein instead of ovarian carcinoma antigen without carrier proteins and adjuvant. Ab(3) could be detected in the sera of immunized mice with 6B11mGM by enzyme-linked immunoadsorbent assay test. Moreover, the fusion protein stimulated proliferation of CD4+ T cell from the spleen of BALB/c mice and proliferation of CD8+ T cell to a lesser degree. Therefore, 6B11mGM probably induces both humoral and cellular immunity against ovarian carcinoma in vivo.     

HPV_(16)L1-E7重组病毒及其重组质粒免疫后小鼠抗体水平动态变化的研究

目的 :研究人乳头瘤病毒 16型L1 E7重组腺病毒 (rAd5HPV16 L1 E7virus)及其重组质粒 (rAd5HPV16 L1 E7plasmid)经不同途径免疫后小鼠抗体水平的动态变化。方法 :用 2 93细胞扩增rAd5HPV16 L1 E7重组病毒 ,同时制备rAd5HPV16 L1 E7重组质粒 ,分别以肌肉注射、腹腔注射、滴鼻和灌胃途径免疫小鼠 ,采用ELISA法测定其血清IgG抗体水平。结果 :rAd5HPV16 L1 E7重组病毒及其重组质粒采用不同途径免疫的BALB/c小鼠血清IgG抗体水平均高于对照组 (P <0 .0 5 ) ,其中以肌注组产生抗体量最多 ,并且出现最早。结论 :rAd5HPV16 L1 E7重组病毒及其重组质粒均能刺激B细胞产生抗体介导的免疫应答 ,免疫途径不同 ,抗体峰值的出现时间亦不同 ,测定抗体的最佳时间亦不同。     

Lectin-binding characteristics of mouse oviduct and uterus associated with pregnancy block by autologous antiprogesterone monoclonal antibody

Oviducts and uteri were removed from BALB/cJ and F1 (CBA/Ca X BALB/cJ) mice at known stages post coitum and following treatment with an antiprogesterone monoclonal antibody (DB3) or a non-specific immunoglobulin (DNP). Thin sections of tissue were prepared and reacted with fluorescent conjugates of a wide range of lectins to determine if saccharide alterations were associated with the pregnancy-inhibiting effect of the DB3 antibody in BALB/c, but not F1, individuals. The ampullary region of the DB3-treated BALB/c mice showed the most marked changes, with an almost total inhibition of lectin binding, particularly for N-acetylglucosamine residues. There was also a reduced affinity for a lectin reactive with N-acetylgalactosamine in the uteri of DB3-treated BALB/c mice, associated with an extended expression during gestation of this saccharide in the proximal region of the oviduct in such mice. These are the first biochemical alterations in reproductive tract epithelia to be associated with the efficacy of the DB3 antibody in preventing pregnancy.     

Superovulation, in vitro fertilization (IVF) and in vitro development (IVD) protocols for inbred BALB/cJ mice in comparison with outbred NMRI mice

Purpose

To study assisted reproductive technology (ART) protocols including superovulation, in vitro fertilization (IVF) and in vitro development (IVD) for BALB/cJ mice in comparison with a common ART protocol for NMRI mice.

Methods

Adult NMRI and BALB/cJ mice were superovulated using a 48?h G-interval. In order to find a more suitable G-interval for the BALB/cJ strain, G-intervals including 44, 46 and 50?h were also examined. Superovulation rates were recorded in all groups. IVF rate of BALB/c oocytes in T6 and mHTF media were compared. IVD rates of BALB/cJ zygotes in mHTF, T6 and G1V₅/G2V₅ media were compared. In addition, IVF and IVD rates of BALB/cJ and NMRI oocytes were compared in T6 medium during IVF?CIVD procedures.

Results

In BALB/cJ mice the highest superovulation rates were observed with 44?C46?h G-intervals. However, with a 48?h G-interval, superovulation rates were significantly lower in BALB/cJ compared to NMRI mice (p?<?0.05). mHTF medium significantly increased in vitro fertilization of BALB/cJ oocytes compared to T6 medium (p?<?0.05). Fertilization rate of NMRI oocytes was significantly higher than BALB/cJ oocytes in T6 medium (p?<?0.05). The BALB/cJ embryo IVD was significantly higher in G1/G2 medium compared to mHTF and T6 media (p?<?0.01).

Conclusions

Superovulation with 48?h G-interval and using T6 during all in vitro procedures produces embryos more efficiently for NMRI mice than for BALB/cJ mice. For BALB/cJ mice, a protocol including superovulation with a 44?C46?h G-interval, using mHTF during IVF and G1V₅/G2V₅ medium during IVD, may improve in vitro embryo production.     

一种多价精子抗原表位肽的设计与合成

目的 :开发可供两性使用的多价合成嵌合肽疫苗。方法 :选择小鼠精子 /睾丸特异性抗原 SP1 7、Cyritestin中特异性氨基酸序列与牛核糖核酸酶非限制性 T细胞表位作为骨架 ,按一定连接方式设计、合成新抗原 3 5肽 ,并与等量弗氏佐剂混悬后免疫雌性 BALB/c小鼠。结果 :在 43 0 A固相自动肽合成仪上成功地合成了 3 5肽 ,并经 HPLC纯化、质谱鉴定证实其纯度达 95 %以上 ;新抗原免疫小鼠后获得的特异性抗血清可识别小鼠、大鼠及人睾丸组织蛋白提取物中相应分子量的蛋白质 ,且血清中特异性 Ig G抗体滴度最高达 1∶ 6 0 0 0 ,阴道粘膜冲洗液中特异性 Ig A抗体滴度最高达 1∶ 3 0 0。结论 :由含有两种特异性精子抗原、具有特异性氨基酸序列的新抗原所制备的抗血清可识别两种天然抗原 ,并在小鼠体内激发出较理想的特异性体液免疫 ,从而为研制适宜于两性的多价抗生育疫苗提供了实验基础     

表达精子抗原受精素β的重组沙门氏菌疫苗株的免疫性避孕效应研究

目的 :探讨表达精子抗原受精素β胞外区的重组沙门氏菌疫苗株用作避孕疫苗的可行性。方法 :应用表达精子抗原受精素β胞外区的两种重组沙门氏菌疫苗株 X46 3 2 (p FEC)和 X45 5 0(p FEC)口服接种雌鼠 ,通过 ELISA方法检测其激发雌鼠产生抗重组受精素 β蛋白的血清 Ig G抗体和阴道分泌物 Ig A抗体滴度变化 ,并观察其诱导的雌鼠免疫性避孕效应情况。结果 :重组 X46 3 2(p FEC)和 X45 5 0 (p FEC)疫苗株不但可激发雌鼠产生高水平血清抗重组受精素 β蛋白和抗菌体LPS Ig G抗体 ,而且在阴道分泌物中可出现一定程度抗重组受精素 β蛋白和抗菌体 LPS Ig A抗体。同时 ,口服免疫后的雌鼠生育能力明显降低 ,两实验组雌鼠妊娠率降低 3 0～ 3 5 %,一胎产仔数量平均降低 6 4 .76～ 6 5 .6 4 %。结论 :表达精子抗原受精素β的重组沙门氏菌疫苗株可在小鼠中诱导免疫避孕效应。     

Antibodies to microbial, leukocyte and organ antigens

Hemagglutinating antibodies to Mycoplasma hominis were present in 30 of 83 infertile, 15 of 40 pregnant and 5 of 20 post-partum females and 20 of 82 infertile males in contrast to only 2 of 21 fertile females and 5 of 25 fertile males. Their presence correlated with sperm antibody detection by TAT in Lab. 4, the immunobead-binding assay of Lab. 1 and the SIT of Lab. 11, but not with other sperm antibody assays. Immunofluorescent antibodies to Chlamydia trachomatis, on the other hand, did not correlate with the incidence of sperm antibodies. Among 305 serum samples tested, 12 were positive for testicular antibodies, 8 had antibodies to kidney, 7 to ovary and 15 to endometrium. A majority of serum samples positive for antibodies to testis and ovary, but not endometrium, reacted against sperm in different assays. Eight of 135 samples tested had antibodies to human leukocyte antigenic HLA-Aw19 (Aw19, A28, A29, A30 and A32) and/or B35 (B35, B5 and B15) complexes. Six of these samples were also positive for sperm antibodies by one or more antibody assays. Cross-reactive antigens may be present in sperm, M. hominis, testis, ovary and leukocytes.     

卵巢癌抗独特型抗体6B11特异性表位肽在体内诱导抗原特异性体液免疫应答

目的探讨卵巢癌抗独特型抗体6B11表位多肽与诱导免疫应答之间的关系。方法采用化学合成的方法，合成抗独特型抗体6B11轻重链可变区的六条CDR区，分别以三种不同浓度免疫BALB／c小鼠。采用ELISA的方法检测免疫鼠血清，进行体内免疫学功能研究。结果6B11抗独特型抗体的六条CDR肽分别加入佐剂免疫小鼠后，其中有两条肽可以诱导出Ab3，分别为重链的CDR2区和轻链的CDR2区。结论这两条肽作为后选肽有可能是6B11抗独特型抗体特异性的CTL表位。     

Effect of immunization on the development of latent ganglionic infection in mice challenged intravaginally with herpes simplex virus types 1 and 2.

Immunization of BALB/c mice with virulent and avirulent strains of HSV-1 resulted in high levels of neutralizing antibody and protected against both the lethal effect of the virus and the development of a latent ganglionic infection when animals were challenged by the intravaginal route. In animals immunized with avirulent strain of HSV-2 and challenged with a high virulent strain of HSV-2, substantial protection against death was observed despite low levels of neutralizing antibody. Nineteen per cent of the survivors, however, developed a latent ganglionic infection. Relatively little protection was observed in mice immunized with HSV-1 and challenged with HSV-2.     

Estrogen promotes B cell activation in vitro through down-regulating CD80 molecule expression

Estrogen is the main female hormone of women. It has diverse effects on cell growth, differentiation and homeostatic functions. Accumulated evidence has indicated that estrogen may regulate multiple immune functions and the immune status of women. However, there is little report on the effect of estrogen on mature B cell functions. In this study, we observed the effect of 17β-estradiol (E2) on the proliferation, apoptosis, antibody production and differentiation of splenic B cells of mice in vitro. Splenocytes of female BALB/c mice were isolated and cultured with E2. E2 treatments decreased the expression of CD80 molecule on splenic B cells but enhanced the total IgG antibody production of splenocyte, without promoting the differentiation of B cells to plasma cells. E2 protected splenic B cells from the serum-deficiency-induced apoptosis but had no influence on the proliferation of B cells. These results suggest that estrogen may promote the activity of B cells through down-regulating the expression of CD80 molecule on B cells.     

Spectrotypic analysis of passively acquired and newly synthesised IgG antibodies in the neonatal and young mouse

Isoelectric focussing with autoradiography has been used to analyse the selective nature of passive transmission of specific anti-Ig allotypic antibodies from the mother to the young mouse, and to study the generation of spectrotypic (clonal) diversity of autologous IgG2a (carrying the paternal inherited Igh-1^b allotype) in BALB/c × SJL/J F1 (Igh-1^ab heterozygote) mice. Transmission of anti-allotypic antibody to neonatal mice was found to be pI restricted, with selection favouring electrophoretically fast IgG. Comparison of the antibody spectrotype in maternal serum, milk and neonatal serum revealed that the pI restriction in transmission operates at the level of the neonatal gut. Analysis of the paternally inherited Igh-1^b IgG2a molecules as they are first synthesised and secreted into the neonatal serum revealed an extensive polyclonality on first detection by the very sensitive focussing assay. it can be deduced that IgV region diversity is generated by IgG2a-synthesising cells prior to, or at the time of, the first secretion of this class of antibody into the serum.     

Maternal alloimmune reactions towards the murine conceptus and graft-versus-host reaction (GVHR). I. Priming for anti-paternal GVHR by gestation

In the H-2 compatible (but minor loci-incompatible) BALB/c-DBA/2 strain combination (both H-2d), intravenous injection of 1.3 X 10(7) BALB/c spleen cells from virgin females into DBA/2 newborn mice less than 18 h old does not result in a significant lethal graft-versus-host reaction (GVHR). A strong GVHR (79% lethal) is induced if the BALB/c donors have been preimmunized to DBA/2. Spleen cells from BALB/c mice pregnant by DBA/2 males are also able to induce a significant, but weaker, GVHR (16% lethal) indicating a cellular priming to paternal antigens by gestation. A significant difference exists between anti-DBA/2 GVH reactivity of spleen cells from primiparous (22% lethal) and multiparous (9% lethal) allopregnant BALB/c mice, indicating that the allogeneic boosters of successive allogestations act more on the target-protective side of immunity than on the target-aggressive one. Sera from allopregnant mice (BALB/c X DBA/2) inhibit the GVHR induced by their own cells, while sera from isopregnant ones (BALB/c X BALB/c) have no effect. Thymectomy performed at 6-wk of age, six weeks before gestation did not significantly modify the maternal reactivity. A similar priming by allogestation in the same strain combination was found for local GVHR (induced in adult F1 hybrids) resulting in higher (+132%, P less than 0.005) stimulation indices and seen to be specific for the paternal strain, the indices induced by the same cells being lower (-35%, P less than 0.05) compared to that induced by cells from virgin BALB/c, when injected into irrelevant F1 hybrids (BALB/c X CBA).     

Assessment of immune responses to H-Y antigen in naturally inseminated and sperm-injected mice using cell-mediated cytotoxicity assays

An in vitro cell-mediated cytotoxicity assay has been used to assess immunity to the male-specific H-Y antigen in female mice after either insemination with male cells at syngeneic natural mating or after injection with syngeneic sperm cell suspensions. Lymphocytes showing specific cytotoxicity for the H-Y antigen could be recovered from both spleen and lymph nodes of female mice injected with sperm. However, insemination of male cells at natural mating did not apparently prime cytotoxic cells against the H-Y antigen in either the spleen or para-aortic lymph nodes draining the uterus of female mice mated once or repeatedly (3-10 X) in the absence of pregnancy. These results are discussed in relation to the factors regulating the immune responsiveness of the female to inseminated antigens.     

Oral immunization with sperm antigens: possible therapy for sperm antibodies?

Young adult male CD-1 mice were given intraperitoneal injections (IP) of saline (controls) and pooled sperm or seminal plasma of two autoimmune infertile men and two nonautoimmune fertile men (n = 40 per treatment). Other mice received only an oral challenge with the same antigens (oral controls; n = 20 per treatment). Three weeks after the booster challenge (day 36), 20 mice in each group were orally immunized with the antigens, whereas the other 20 were not (IP controls). Cytotoxic antibody titers (immunoglobulin M) to human sperm were significantly higher in mice IP immunized with sperm or seminal plasma from autoimmune infertile men or orally immunized with autoimmune men's sperm, in contrast to the controls. Oral challenge with sperm or seminal plasma of autoimmune infertile men after the IP immunization with the same resulted in significantly decreased cytotoxic sperm antibody titers (P less than 0.001 versus oral or IP controls in sperm immunization; P less than 0.001 versus IP controls in seminal plasma immunization). Fertility was unaffected by any mode of immunization. It is concluded that, in mice, sperm and seminal plasma antigens from autoimmune infertile men are more immunogenic than those from nonautoimmune fertile men, and oral challenge with the former after an IP establishment of cytotoxic sperm immunity desensitizes the immune mice. These findings may have practical implications in the diagnosis and immunotherapy of infertile men with cytotoxic sperm antibodies.     

Adhesion formation and interanimal variability in a laparoscopic mouse model varies with strains

Adhesion formation after laparoscopic surgery was evaluated in mice of different strains. More adhesions were observed in Swiss, NMRI, and BALB/c mice, with less interanimal variability in BALB/c mice. These data point to genetics effects on adhesion formation, which open new insights in its pathogenesis and indicate the importance of a careful strain selection for animal studies.     

Passive immunization with antisperm monoclonal antibody ZAP-7 increases preimplantation embryo mortality in the mouse

Objective: To evaluate the effect of VIC-1 and ZAP-7 antihuman sperm monoclonal antibodies on in vivo fertility in the mouse.

Design: A randomized blinded study using a mouse model.

Setting: University-based laboratory.

Animals: B6CBAF1 mice (n = 6 per experimental group).

Intervention(s): Antisperm antibodies were administered intravaginally to female mice before mating. Control mice received no treatment, saline, or nonspecific antibodies. Number and viability of preimplantation embryos were determined by microscopic observation. Mouse sperm, oocytes, and normal preimplantation embryos were used in indirect immunofluorescence assays with antisperm antibodies. The effect of antibody treatment on sperm motility and vitality was evaluated.

Main Outcome Measure(s): Antigen expression, sperm motility and vitality, number and viability of embryos.

Result(s): ZAP-7 antibody recognizes a sperm antigen expressed in zygotes and early preimplantation embryos. Passive immunization with ZAP-7 increases embryo mortality significantly (more than 40% above controls). Passive immunization with VIC-1 has no deleterious effect.

Conclusion(s): ZAP-7 monoclonal antibody disrupts fertilization and embryogenesis in the mouse.     

Increased severity of Candida vaginitis in BALB/c nu/nu mice versus the parent strain is not abrogated by adoptive transfer of T cell enriched lymphocytes

The role of the host immune system in combating candidal infections in the vagina is poorly understood. A murine model of Candida vaginitis was used to elucidate the role of T cells in a candidal infection. Athymic BALB/c nu/nu mice or normal BALB/c mice were induced into estrus and then infected with 1 x 10(6) Candida albicans intravaginally. The infection was monitored over 1 week. Samples from blood, small intestine, tongue, kidney, spleen, liver, uterus and vagina were tested for recoverable C. albicans. Histology of the vagina was assessed for both inflammation and extent of infection. Results indicated that the BALB/c nu/nu mice had similar levels of vaginal yeast load to the normal BALB/c mice. In 25-30% of nude mice Candida was also recovered from extra vaginal sites (kidney, liver, small intestine), however, extra vaginal dissemination was not observed in any normal BALB/c animals. Histologically, both the nu/nu and control BALB/c had similar levels of vaginal inflammation; however, the nu/nu mice had more florid fungal growth in the vaginal epithelium. Adoptive transfer of either immune or non-immune BALB/c T cells into nude mice had no affect on either infection or vaginal inflammation. Immunohistochemical staining of vaginal tissues from normal BALB/c mice or nude mice adoptively transferred with either immune or non-immune T cells with anti-CD3 monoclonal antibody revealed no significant difference between groups in the numbers of CD3+ vaginal T cells. However, in mice receiving either immune or non-immune T cells no yeast was recovered from any tissues except the vagina. These data show that T cells have a limited role in protecting the vagina from C. albicans infection.     

自身免疫性卵巢功能衰退小鼠体液免疫和细胞免疫的测定

目的探讨自身免疫性卵巢功能衰退者体液免疫和细胞免疫的变化。方法在建立自身免疫性卵巢功能衰退小鼠动物模型基础上,测定血自身抗卵巢抗体（ＡＯＡ）和Ｔ细胞亚群变化。结果自身免疫性卵巢功能衰退小鼠辅助性诱导性Ｔ细胞（ＣＤ＋４）无明显变化,而细胞毒性抑制性Ｔ细胞（ＣＤ＋８）百分比明显下降（Ｐ＜０．０１）,ＣＤ４／ＣＤ８比率明显增加（Ｐ＜０．０５）;血清ＡＯＡ阳性,并导致卵巢组织病理性损伤,性激素产生减少,生育率降低。结论Ｔ细胞亚群比例失调、Ｂ细胞功能增强,是导致自身免疫性卵巢功能衰退的免疫病理基础