[Application of the two-step sentinel lymph node biopsy with double-tracer in early staged endometrial cancer].

Objective: To explore the feasibility and clinical value of sentinel lymph node (SLN) biopsy through cervix-uterine combined two-step injection with two tracers in patients with early stage endometrial cancer. Methods: From July 2019 to April 2021, a total of 73 patients, aged (54.2±3.3) year, who were preoperatively diagnosed as stage Ⅰ-Ⅱ endometrial cancer (including 56 low-risk patients and 17 medium-high risk patients) in Affiliated Hospital of Qingdao University were selected. According to the different sites of tracer injection, the patients were randomly divided into three groups: cervical injection group (25 cases): 1 ml of nano-carbon was used to inject at 3 and 9 o'clock in the cervix; uterine injection group (21 cases): the magnetic resonance imaging examination was performed to determine the location of the lesion, and 4 ml of methylene blue was injected into the uterine body at 2 sites where the lesion was located; combined injection group (27 cases): cervical injection of nano-carbon (1 ml) combined with uterine injection of methylene blue (4 ml). The SLN in all patients were identified under laparoscopy, removed, and followed by frozen pathological examination. Pathological ultra-staging was performed if the postoperative pathological outcome of SLN was negative. The total detection rate of SLN, bilateral pelvic SLN detection rate, sensitivity, negative predictive value, and location of SLN in each group were calculated and compared. Results: (1) In 73 patients with endometrial cancer, the overall detection rate of SLN was 88% (64/73), the detection rate of bilateral pelvic SLN was 67% (49/73), and the detection rate of para-aortic SLN was 49% (36/73). The overall detection rate of SLN (71%, 15/21) and bilateral pelvic SLN (43%, 9/21) in the intrauterine injection group were significantly lower than those in the cervical injection group [92% (23/25), 76% (19/25), respectively] and the combined injection group [96% (26/27), 78% (21/27), respectively; all P<0.05]; the detection rate of para-aortic SLN in the cervical injection group (28%, 7/25) was significantly lower than those in the intrauterine injection group and combined injection group [52% (11/21) and 67% (18/27), respectively; both P<0.05]. Among 73 cases with endometrial cancer, 9 had lymph node metastasis confirmed by postoperative pathological examination, 8 of them had lymph node metastasis detected by SLN and 1 had no lymph node metastasis detected by SLN, with a total sensitivity of 89% and a negative predictive value of 98%. The sensitivity and negative predictive value of cervical injection group and combined injection group were 100%, while the sensitivity and negative predictive value of intrauterine injection group were 67% and 95%. Among 56 low-risk patients, only one patient with lymph node metastasis was confirmed by postoperative pathology by SLN detection, and the metastasis rate was 2% (1/56), and the sensitivity and negative predictive value were 100%. Lymph node metastasis was confirmed in 8 of 17 patients (8/17) with a sensitivity of 88% and a negative predictive value of 90%. (2) A total of 459 SLN were detected in 73 endometrial cancer patients, with the highest proportion of external iliac (33.3%, 153/459).The obturator foramen was 25.3% (116/459), para-aortic 19.6% (90/459), iliac 12.0% (55/459), and presacral 9.8% (45/459). The proportion of para-aortic SLN in the cervical injection group was 12.4% (21/169), which were significantly lower than that in the intrauterine injection group and the combined injection group [27.4% (26/95) and 22.1% (43/195), respectively; both P<0.05]. (3) Pathological super-staging results: among 64 patients with negative SLN routine paraffin pathology, 4 cases of lymph node micro-metastases and 1 case of isolated tumor cell metastasis were detected, and the SLN micro-metastases rate was 8% (5/64), including 2 cases of low-risk patients and 3 cases of medium-high risk patients. Conclusions: SLN biopsy has high sensitivity and negative predictive value in patients with early endometrial cancer and could be used as an alternative to systematic lymph node dissection in low-risk patients. The SLN mapping through cervical-uterine combined injection could further improve the detection rate effectively and avoid the missed detection of positive para-aortic lymph node, especially for high-risk patients or patients with fundal tumor involvement.

[Effects of ascites derived exosomes on the stemness and invasion ability of ovarian cancer stem-like cell].

Objective: To investigate the effects of ovarian cancer ascites-derived exosomes on the stem cell properties and invasion ability of ovarian cancer stem-like cell (OCS-LC). Methods: (1) A2780 cells were induced into OCS-LC in serum-free medium, and authenticating their stem-like properties by sphere-forming test, differentiation test and CD133 marker detection. (2) Exosomes from ascites and A2780 cell were extracted by ultracentrifugation, then authenticating them. The morphology of exosomes was observed by the transmission electron microscope, exosome particle size was measured by nanoparticle tracking analysis (NTA). The expressions of heat shock protein 70 (HSP-70), CD63 and CD9 were detected by western blot. (3) The exosomes from ovarian cancer ascites and tumor cell supernate were co-cultured with OCS-LC. The groups were divided into control group, ascites-derived exosomes (ADE) group (ADE+OCS-LC group), and cells-derived exosomes (CDE) group (CDE+OCS-LC group). The sphere-forming ability was evaluated by sphere-forming cycle, maximum sphere diameter and sphere-forming rate of each group; the expression of CD133 was detected by immunofluorescence staining under microscope; quantitative real-time (qRT)-PCR was used to detected the expression levels of octamer-4 (Oct-4), Nanog mRNA of the signature genes in the stem cells of each group; the metastasis ability of each group was measured by transwell assay. Results: (1) Identification of OCS-LC: sphere-forming experiment showed that the suspension of OCSC single cells was grown in serum-free medium in secondary sphere-forming. Differentiation function experiment showed that OCS-LC were differentiated into adherent A2780 cells by changing the growth mode in serum containing medium. Flow cytometry showed that the proportion of CD133 positive (CD133+) cells in OCS-LC group was (18.9±0.9)%, significantly higher than that of control group (0.6±0.5)% (t=38.570, P<0.01). (2) Under transmission electron microscope, clear lipid bilayer structure was observed in ADE and CDE, and one side presented a concave hemispheric or cup like structure. NTA showed that the diameter of exosomes mainly ranged from 30 to 100 nm, with an average diameter of 67.2 nm. Western blot analysis showed that the specific protein molecules HSP-70, CD63 and CD9 were positive. (3) Three groups' OCS-LC could continue to grow into spheres, and the group of ADE+OCS-LC showed two growth modes, most of the cells continued to grow into spheres, while a small part of cells grew in adherent differentiation. The sphere-forming rate of OCS-LC in the control group, ADE+OCS-LC group, and CDE+OCS-LC group were (1.05±0.20)%, (4.15±0.10)%, and (10.45±0.25)%, the sphere-forming cycle of OCS-LC in the three groups were (15.3±1.5), (10.3±0.6), and (6.7±0.6) days, and the maximum diameters of OCS-LC were (100.3±3.2), (145.2±5.1) and (170.0±2.1) µm, respectively. And the differences were statistically significant (all P<0.05). After co-culture of exosomes with OCS-LC, the sphere-forming ability of cells in the group of CDE+OCS-LC was prior to ADE+OCS-LC group (all P<0.05). Immunofluorescence staining showed that the number of CD133 green fluorescent chromophore cells in OCS-LC groups [(46.2±2.1)%, (58.4±2.2)%] was significantly higher than that in the control group [(26.6±1.5)%] after the addition of exosomes in co-culture, the positive rate of CD133 was higher than that in the control group(F=187.588, P<0.05). The qRT-PCR results showed that the expression levels of Oct-4 mRNA in ADE+OCS-LC and CDE+OCS-LC groups were 3.46±0.24, 4.03±0.31, compared with that in control group (1.04±0.12), the differences were statistically significant (F=134.932, P<0.05). The mRNA expression levels of Nanog were 1.57±0.32, 2.66±0.15, which were significantly higher than that in the control group (1.00±0.07), and the differences were statistically significant (F=49.329, P<0.05). And the expression of both in CDE+OCS-LC group increased more significantly than ADE+OCS-LC group (all P<0.05). The number of invasive cells in the three groups of OCS-LC were: control group 30±5, ADE+OCS-LC group 102±4, CDE+OCS-LC group 210±7, and there were statistically significant differences among three groups (F=820.800, P<0.05). Compared with the control group, the number of invaded cells in the co-culture group were significantly increased (P<0.05), and the CDE+OCS-LC group had the higher cell invasion ability then the ADE+OCS-LC group (t=23.202, P<0.05). Conclusions: Exosomes derived from ovarian cancer ascites could enhance and maintain the stemness of OSC-LC, and promote the invasion of tumor cells. Moreover, CDE is superior to ADE.

Amelogenin domain-containing NlChP38 is necessary for normal ovulation in the brown planthopper.

The brown planthopper (BPH), Nilaparvata lugens, is a major threat to rice production. The eggshell plays an important role in insect reproduction. The constituents and formation process of BPH eggshells remains largely unknown. Here, we report a novel eggshell-associated protein, NlChP38, containing an amelogenin domain, that is essential for normal ovulation in the BPH. NlChP38 is specifically expressed in the follicular cells from egg chambers at both RNA and protein levels. RNA interference of NlChP38 resulted in oocytes with loose and thin eggshell structure and caused ovulation difficulties. Immunofluorescence localization showed NlChP38 is deposited between follicular cells and oocytes during late choriogenesis. These results indicate that NlChP38 plays an important role in eggshell formation and could be a potential target for RNA interference control of the BPH.

Identification and functional analysis of a novel chorion protein essential for egg maturation in the brown planthopper.

In insect eggs, the chorion has the essential function of protecting the embryo from external agents during development while allowing gas exchange for respiration. In this study, we found a novel gene, Nilaparvata lugens chorion protein (NlChP), that is involved in chorion formation in the brown planthopper, Nilaparvata lugens. NlChP was highly expressed in the follicular cells of female adult brown planthoppers. Knockdown of NlChP resulted in oocyte malformation and the inability to perform oviposition, and electron microscopy showed that the malformed oocytes had thin and rough endochorion layers compared to the control group. Liquid chromatography with tandem mass spectrometry analysis of the eggshell components revealed four unique peptides that were matched to NlChP. Our results demonstrate that NlChP is a novel chorion protein essential for egg maturation in N. lugens, a hemipteran insect with telotrophic meroistic ovaries. NlChP may be a potential target in RNA interference-based insect pest management.

[Expression and function of long intergenic non-protein coding RNA-regulator of reprogramming in high-grade ovarian serous cancer].

Objective: To investigate the expression of long intergenic non-protein coding RNA-regulator of reprogramming (Linc-ROR) in high-grade ovarian serous cancer, and explore the relationship between Linc-ROR expression and biological function of high-grade ovarian serous cancer. Methods: A total of 34 high-grade ovarian serous cancer tissue samples and 19 normal fallopian tube tissue samples were collected between June 2014 and February 2016. Real-time reverse transcription (RT)-PCR was used to detect the Linc-ROR mRNA expression in different samples. The relationship between Linc-ROR expression level and ovarian cancer International Federation of Gynecology and Obstetrics (FIGO) stage, lymph node metastasis was analyzed. Constructed Linc-ROR small interference RNA (siRNA) and pIRES2-EGFP-Linc-ROR plasmid, then Linc-ROR siRNA and pIRES2-EGFP-Linc-ROR plasmid were respectively transfected into SKOV3 cells. Cell proliferation, migration and invasion ability were assessed by cell counting kit-8 (CCK-8), wound healing assay and transwell invasion assay. Results: (1) The expression level of Linc-ROR mRNA was significantly higher in high-grade ovarian serous cancer than normal fallopian tube tissues (4.31± 0.38 vs 1.03 ± 0.21; t=25.842, P<0.01). With the progression of FIGO stages, the expression of Linc-ROR was increased (F=95.702, P<0.01), and it was associated with lymph node metastasis (t=7.397, P<0.01). (2) The results of RT-PCR showed that the expression level of linc-ROR in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (0.30 ± 0.11 vs 1.02 ± 0.10; t=15.269, P<0.01). The expression level in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (8.90± 0.45 vs 1.03±0.17; t=21.934, P<0.01). The CCK-8 assay showed that when the cells were cultured for 3, 4, 5 and 6 days, the A value in Linc-ROR-i group was significantly lower than that in Linc-ROR-NC-i group (P< 0.05). And the A value in Linc-ROR-p group was significantly higher than that in Linc-ROR-NC-p group (P< 0.05). Wound healing assay showed that, after 48 hours incubation, migration rate of cells in Linc-ROR-i group was significantly less than that in the Linc-ROR-NC-i group [(52±4)% vs (67±5)%; t=5.720, P< 0.01]. The migration of cells in Linc-ROR-p group was significantly greater than that in the Linc-ROR-NC-p group [(84±4)% vs (66±4)%; t=7.330, P <0.01]. Cell transwell invasion assay showed that, after 48 hours of incubation, the number of invasive cells in Linc-ROR-i group was lower than that in Linc-ROR-NC-i group (74 ± 3 vs 104 ± 3; t=15.810, P<0.01). And the number of invasive cells in Linc-ROR-p group was higher than that in Linc-ROR-NC-p group (217 ± 4 vs 108 ± 5; t=38.060, P<0.01). Conclusion: Highly expressed Linc-ROR could enhance the proliferation, migration and invasion ability of high-grade ovarian serous cancer cells, which may be one of the important molecules in the occurrence and development, invasion and metastasis of high-grade ovarian serous cancer.

Immune responses to Ro60 and its peptides in mice. I. The nature of the immunogen and endogenous autoantigen determine the specificities of the induced autoantibodies.

Anti-Ro60 autoantibodies are found in a variety of autoimmune disorders including systemic lupus erythematosus (SLE), Sjögren's syndrome, primary biliary cirrhosis, and active hepatitis. They are the most prevalent autoantibodies in normal individuals and in asymptomatic mothers of infants afflicted with neonatal lupus. In the present study, immune responses to recombinant human Ro60 (rhRo60) and recombinant mouse Ro60 (rmRo60) and selected Ro60 peptides in non-SLE-prone mice were investigated. Multiple T and B cell epitopes were identified in Ro60. Immunizations with either xenogeneic or autologous Ro60 induced autoantibodies to a diverse group of autoantigens. In addition to La and Ro52, proteins in the small nuclear ribonucleoprotein (snRNP) particles such as SmA, SmB, SmD, and 70-kD U1-RNP were unexpectedly identified as targeted antigens. In the studies involving synthetic Ro60 peptides, both human and mouse Ro60316-335 peptides, which differ in three amino acids, were found to contain dominant cross-reactive T cell determinants. Immunizations with these peptides induced autoantibodies to Ro60, La, SmD, and 70-kD U1-RNP without autoantibodies to Ro52, SmA, or SmB. With human Ro60316-335 as the immunogen, additional autoantibodies reactive with the Golgi complex were found. In contrast to the immunodominance of both human and mouse Ro60316-335 peptides, the T cell determinant in human Ro60441-465 was dominant, whereas that in the mouse peptide was cryptic. Immunization with human Ro60441-465 induced primarily anti-peptide Abs. Mouse Ro60441-465 failed to induce an antibody response. These results show that both the nature of the immunogen and the immunogenicity of the related endogenous antigen are important in determining the specificities of the autoantibodies generated. They have significant implications for proposed mechanisms on the generation of complex patterns of autoantibodies to a diverse group of autoantigens in SLE patients.

Structural and functional consequences of alveolar cell recognition by CD8(+) T lymphocytes in experimental lung disease.

CD8(+) T cells infiltrate the lung in many clinical conditions, particularly in interstitial lung disease. The role(s) that CD8(+) T cells might be playing in the pathogenesis of inflammatory lung disease is unclear at present, as is the direct contribution of CD8(+) T cell effector activities to lung injury. This report describes a transgenic model used to evaluate the impact, on respiratory structure and function, of CD8(+) T lymphocyte recognition of a target antigen expressed endogenously in alveolar epithelial cells. We found that adoptive transfer of cloned CD8(+) cytotoxic T lymphocytes (CTLs) specific for an alveolar neo-antigen (influenza hemagglutinin) leads to progressive lethal injury in transgenic mice, which dramatically affects lung structure and function. Transgenic recipients of CD8(+) CTLs exhibited tachypnea and progressive weight loss, becoming moribund over a period of several days. Concomitantly, the animals developed a progressive interstitial pneumonitis characterized initially by lymphocytic infiltration of alveolar walls and spaces, followed by an exuberant mononuclear cell infiltration that correlated with restrictive pulmonary mechanics and a progressive diffusion impairment. These results indicate that antigen-specific CD8(+) T cell recognition of an alveolar epithelial "autoantigen" is, in and of itself, sufficient to trigger an inflammatory cascade that results in the histological and physiological manifestations of interstitial pneumonia.

Autoimmune disease of the ovary induced by a ZP3 peptide from the mouse zona pellucida.

We describe a novel experimental system in mice for the study of ovarian autoimmune disease, a condition encountered in women with premature ovarian failure. The ovarian autoimmune disease is induced in B6AF1 mice by a 15-amino acid peptide (Cys-Ser-Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln-Ile-His-Gly-Pro-Arg) from mouse ZP3, the sperm-binding component of the zona pellucida that surrounds growing and mature oocytes. Whereas the peptide induces both T cell and antibody responses, adoptive transfer of CD4+ T cell lines derived from affected animals causes oophoritis without observable antibodies to the zona pellucida peptide. The primacy of the T cell response in the pathogenesis of disease is further substantiated by defining oophoritogenic peptides as small as eight amino acids (Asn-Ser-Ser-Ser-Ser-Gln-Phe-Gln) that do not elicit an antibody response to the full-length ZP3 peptide. The identification of a well characterized peptide as a causative agent of autoimmune oophoritis should facilitate understanding of the pathogenesis of this T cell-mediated autoimmune disease. Because the proteins of the zona pellucida are conserved among mammals (the mouse and human ZP3 proteins are 67% identical), this murine model may lead to better understanding of the pathogenesis of human autoimmune oophoritis.

Autoimmune ovarian disease: mechanism of disease induction and prevention.

Investigation of the versatile models for autoimmunity of the ovary and other selected organs has contributed to our understanding of the following aspects of autoimmunity: the mechanism of T cell molecular mimicry; T-->B epitope spreading, as a basis for autoantibody diversification, and as a link between organ-specific and systemic autoimmunity; the localization of genetic loci potentially influencing multiple autoimmune diseases; and the elucidation of regulatory T cells as a component of physiological self tolerance.

Mechanism of ovarian autoimmunity: induction of T cell and antibody responses by T cell epitope mimicry and epitope spreading.

Autoimmune diseases are often manifested as organ inflammation with loss of function, and detectable autoreactive T cell and autoantibody responses. In the proper genetic context, we have shown that these parameters of autoimmunity can result from a single pivotal event: the induction of a strong and persistent T cell response for a foreign or unrelated self peptide that mimics the target self peptide. This may apply to organ-specific and systemic autoimmunity, independent of whether the tissue inflammation results from T cell immune mechanism or antibodies. T cell peptide mimicry, through sharing of critical residues or by a less defined mechanism, can result in autoimmune disease. Once triggered, the helper T cell response leads rapidly to a concomitant autoantibody response spreading to distant B cell determinants of the self protein antigen. Evidently, with T cell help, endogenous antigens can stimulate B cells to provoke a functional autoantibody response against conformational antigenic determinants. These findings are based on recent studies on a novel autoimmune ovarian disease model induced by a self peptide with well-defined T and B cell epitopes. However, studies reported on systemic lupus erythematosus models have shown that similar events may result in autoantibody response in systemic autoimmunity.

Contraceptive vaccine assessment based on a murine ZP3 mini-autoantigen.

A summary is presented of published and some unpublished observations from studies on the immunological response of mice to a 13-mer peptide of the murine ovarian zona pellucida glycoprotein ZP3. The findings have the following implications for the design of immunocontraceptive vaccines. To be reversible, a ZP3 vaccine must not contain pathogenic T cell epitopes of ZP3, but contraception without autoimmune oophoritis may be feasible. The immune response to the ZP3 mini-autoantigen is highly variable among inbred mouse strains, suggesting that a single oophoritogenic peptide would not achieve irreversible contraception in an outbred population. The discovery of antigen mimicry at the level of T cell peptide has thrown doubt on the validity of current strategy in detecting relevant self-antigens that might cross react with vaccine immunogens and on the feasibility of fully predicting the cross-reactive autoimmunogenic potential of a peptide or polypeptide vaccine antigen. Autoantibodies directed against epitopes outside the ZP3 mini-autoantigen, produced by immunization with the pure T cell epitope, react with high affinity, with native zona pellucida, and may be useful in identifying B cell epitopes in ZP3.

[Studies on mutation of isoniazid resistant genes in M. tuberculosis].

OBJECTIVE: To understand mutation of isoniazid-resistant genes in M. tuberculosis clinical isolates, and to develop the method for rapid detection of drug resistance. METHOD: Analyzing the katG genes in 30 M. tuberculosis clinical isolates with PCR-SSCP(single stranded conformation polymorphism) and PCR-DS (direct sequencing) techinques. RESULTS: With the reference of M. tuberculosis strain H37 Rv was used as control, all isoniazid-sensitive isolates and 4 isoniazid-resistant isolates displayed ab normal PCR-SSCP and PCR-DS patterns. But of 12 isoniazid-resistant isolates, 4(33.4%) isolates displayed abnormal katG PCR-SSCP pattern, 8 (46.7%) isolates displayed abnormal katG PCR-SSCP pattern, 5(41.7%) isolates were missense mutation at codon 94, 3(25%) isolates were cosense mutation at codon 96. CONCLUSIONS: Resistance to isoniazid in most M. tuberculosis isolates is due to the mutations on katG genes. PCR-SSCP techniques could be used as a method for rapid detection of isoniazid-resistance in M. tuberculosis.

Physical biochemical properties of IgM from a teleost fish.

Teleost fish are able to produce IgM class antibody, as are other vertebrates. When the teleost fish Oreochromis niloticus was immunized with bovine serum albumin (BSA), it produced antibody to BSA with an average avidity of 7.4 x 10(8)/M. Thus, dissociation of antigen-antibody complexes only occurred at conditions of < pH 2.5, > pH 11, > 4M NaI or > 4M urea, demonstrating high stability of the complex. Western blot analyses further showed the high specificity of the antibody to BSA. In contrast to mammals, when the fish was challenged with multiple protein antigens, it produced antibody only to the major component but not to others. The antibody generated to a specific antigen accounted for up to 1.1% of whole serum protein or 7.0% of whole immunoglobulin. We conclude (1) the systemic antibody response in teleost fish may be an 'all or nothing' response, which is different from that in mammals; (2) the quality (specificity and affinity) of the antibody produced is similar to that of mammals. The findings not only reveal a quite different strategy of immune response in fish, but also raise the possibility of technical application.

Cyclin B in fish oocytes: its cDNA and amino acid sequences, appearance during maturation, and induction of p34cdc2 activation.

Under the influence of maturation-inducing hormone (MIH) secreted from follicle cells, oocyte maturation is finally triggered by maturation-promoting factor (MPF), which consists of a homolog of the cdc2+ gene product of fission yeast (p34cdc2) and cyclin B. Two species of cyclin B clones were isolated from a cDNA library constructed from mature goldfish oocytes. Sequence comparisons revealed that these two clones are highly homologous (95%) and were found to be similar to Xenopus cyclin B1. Using monoclonal antibodies against Escherichia coli-produced goldfish cyclin B and the PSTAIR sequence of p34cdc2, we examined the levels of cyclin B and p34cdc2 proteins during goldfish oocyte maturation induced in vitro by 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17 alpha, 20 beta-DP), a natural MIH in fish. Protein p34cdc2 was found in immature oocyte extracts and did not remarkably change during oocyte maturation. Cyclin B was not detected in immature oocyte extracts and appeared when oocytes underwent germinal vesicle breakdown. Cyclin B that appeared during oocyte maturation was labelled with [35S]methionine, indicating its de novo synthesis. Introduction of E. coli-produced cyclin B into immature oocyte extracts induced p34cdc2 (MPF) activation. Although the possibility that immature goldfish oocytes contain an insoluble cyclin B is not completely excluded, these results strongly suggest that 17 alpha, 20 beta-DP induces oocytes to synthesize cyclin B, which in turn activates preexisting p34cdc2, forming active MPF.

Rapid induction of autoantibodies by endogenous ovarian antigens and activated T cells: implication in autoimmune disease pathogenesis and B cell tolerance.

Animals immunized with nuclear antigenic peptides produce autoantibodies to distant antigenic sites and neighboring Ags within a multimolecular complex. This has led to the hypothesis that induction of autoantibodies in systemic autoimmune diseases might be triggered by a T cell epitope. We have investigated the T to B epitope spreading phenomenon based on the murine autoimmune oophoritis model. Mice immunized with a ZP3 T cell peptide spontaneously produced amplified autoantibodies (amAb) against linear ZP3 B cell epitopes outside the peptide immunogen. Each ZP3 B cell peptide, chimerized to a foreign promiscuous T cell epitope, elicited Ab to the peptide within the native ZP3 molecule. Mice with amAb often had no oophoritis; but more importantly, bilateral ovariectomy 1 day before ZP3 T epitope injection inhibited the induction of the amAb response, whereas ovariectomy 2 to 4 days after immunization was not inhibitory. Because endogenous ovarian Ag depletion before detectable ZP3 T cell response (day 5) and oophoritis (day 7) failed to prevent the amAb response, the autoantibodies are likely stimulated by endogenous ZP3 Ags present outside the normal ovaries. AmAb, of only the IgG class, appeared on day 7; this was 2 to 3 days after detectable T cell response, and 5 to 6 days before A response to the T cell peptide immunogen. The rapid, class-switched amAb response indicates that B cells in female mice are not tolerant to self ovarian Ag and they may normally be primed by ZP3. As evidence for their pathogenic potentials, amAb were produced in response to oophoritogenic, nonovarian T cell peptides that mimic ZP3; moreover, an excellent correlation existed between amAb titers and fertility reduction.

Antibody responses and infertility in mice following oral immunization with attenuated Salmonella typhimurium expressing recombinant murine ZP3.

Ovarian ZP3, the primary sperm receptor, is a major glycoprotein of mouse zona pellucida (ZP). Because antibodies raised against ZP3 block sperm-egg interaction, ZP3 has been considered a candidate immunogen in the development of a contraceptive vaccine. This study explored the possibility of using an attenuated Salmonella typhimurium vaccine strain expressing recombinant ZP3 to elicit an antibody response and infertility in mice. A cDNA sequence generated by the polymerase chain reaction encoding 342 amino acid residues (23-364) of the mouse (m)ZP3 was cloned into an Asd+ vector. An avirulent Salmonella vaccine strain stably expressed the ZP3 polypeptide and colonized the internal organs of mice after oral inoculation. Oral immunization of female BALB/c mice with the recombinant Salmonella vaccine strain expressing mZP3 induced significant levels of anti-native ZP IgG antibodies in serum and IgA antibodies in vaginal secretions. The IgG antibodies thus induced also bound to ZP in vivo. When mated with males, 3 of 6 females immunized with the recombinant Salmonella were infertile. In contrast, none of the mice that received Salmonella containing the vector plasmid produced antibodies to ZP and all were fertile. No ovarian inflammation was observed in the immunized mice at autopsy. The results suggest a potential oral contraceptive vaccine to control populations of rodent vectors of disease and to induce reversible infertility in humans.

Tolerance mechanism in experimental ovarian and gastric autoimmune diseases.

Neonatal splenocytes, neonatal thymocytes, or phenotypically mature adult thymocytes, transferred from normal BALB/c mice to syngeneic athymic nu/nu (or SCID) mice, led to autoimmune oophoritis and autoimmune gastritis, with corresponding serum autoantibodies, in the recipients. The overall disease incidence was 73%; the pathology ranged from mild to severe, with complete loss of ovarian follicles and gastric parietal cells. CD4+ neonatal spleen cells and CD4+ CD8- adult thymocytes were required for autoimmune disease induction. Adult spleen cells did not elicit disease, but they prevented disease when co-transferred with neonatal spleen cells. However, in confirmation of an earlier report by Sakaguchi et al., (J. Exp. Med. 161:72, 1985), a subset of adult splenic T cells expressing a low level of CD5 molecules elicited similar autoimmune diseases. Thus, self-reactive T cells responsible for autoimmune disease of the stomach and ovary are not effectively deleted in the thymus, and they exist in the peripheral lymphoid organs of normal mice. We conclude that the functional expression of the self-reactive T cells is ontogenetically regulated; whereas T cells in the neonatal mice readily elicited autoimmune diseases in nu/nu recipients, regulatory cells may render self-reactive T cells in the normal adults unresponsive.

Retargeting T cell-mediated inflammation: a new perspective on autoantibody action.

To understand the pathogenesis of organ-specific autoimmune disease requires an appreciation of how the T cell-mediated inflammation is targeted, and how the organ function is compromised. In this study, autoantibody was documented to influence both of these parameters by modulating the distribution of T cell-mediated inflammation. The murine autoimmune ovarian disease is induced by immunization with the ZP3330-342 peptide of the ovarian zona pellucida 3 glycoprotein, ZP3. Passively transferred or actively induced Ab to ZP3335-342 bound to the zona pellucida in the functional and degenerative ovarian follicles, and the ovaries remained histologically normal. Transfer of ZP3330-342 peptide-specific T cells targeted the degenerative follicles and spared the functional follicles, and the resultant interstitial oophoritis was associated with unimpaired ovarian function. Unexpectedly, the coexistence of ZP3330-342 peptide-specific T cells and zona-bound autoantibody led to a dramatic translocation of the ovarian inflammation to the growing and mature ovarian follicles, with destruction of the ovarian functional unit. Ab retargeted both Th1-induced mononuclear inflammation and Th2-induced eosinophilic inflammation, and retargeting was induced by murine and rat polyclonal Abs to multiple distinct native B cell determinants of the zona pellucida. Therefore, by reacting with the native determinants in tissue Ag, Ab alters the distribution of T cell-mediated inflammation, and results in destruction of the functional units of the target organ. We propose that this is a clinically important and previously unappreciated element of Ab action in autoimmune disease.

Altered target organ. A mechanism of postrecovery resistance to murine autoimmune oophoritis.

Experimental murine autoimmune oophoritis, a model of human premature ovarian failure, is induced by immunization with a peptide of the ZP3 glycoprotein from mouse zona pellucida (ZP3(330-340)) in CFA. The ovarian pathology is mediated by ZP3-specific, CD4+ T cells, and not by Abs. We now show that mice recovered from autoimmune oophoritis in 4 mo, as characterized by regression of ovarian inflammation. Recovery was associated with disease resistance upon rechallenge with ZP3(330-340) in CFA. Oophoritis resistance was not explicable by immunosuppressive effect of CFA priming, nor by suppression of pathogenic T cells. ZP3-specific, proliferative T cell response could be detected, and a ZP3-specific, IFN-gamma-producing pathogenic T cell line was derived readily from the recovered mice by in vitro stimulation with the ZP3(330-340) peptide. Moreover, recovered mice, when challenged with ZP3(330-340) in CFA, produced Abs of IgG class to the ZP3(330-340) peptide. Suppressor T cells are not readily demonstrable. Most importantly, oophoritis occurred in normal ovaries implanted under the renal capsule of the recovered mice. That oophoritis developed in the implanted ovaries, but spared the endogenous ovaries, further indicates that the latter is refractory to oophoritis. Disease resistance of the ovaries is not explicable by limitation of accessible target Ags. When mated, recovered mice were fertile and produced normal litters; and, as recipients of a ZP3-specific T cell line, their ovaries developed oophoritis. We conclude that altered local environment of the target organ following autoimmune disease recovery can contribute to the complex disease-resistant state.

Glomerulonephritis induced by recombinant collagen IV alpha 3 chain noncollagen domain 1 is not associated with glomerular basement membrane antibody: a potential T cell-mediated mechanism.

Glomerulonephritis is believed to result commonly from Ab-mediated glomerular injury. However, Ab-associated mechanisms alone cannot explain many cases of human glomerulonephritis. We developed a rat model of human anti-glomerular basement membrane (GBM) disease to investigate T cell and Ab response, and their associations with the disease. A single immunization of highly denatured recombinant mouse collagen IV alpha3 chain noncollagen domain 1 (rCol4alpha3NC1) induced severe glomerulonephritis in 100% of Wistar Kyoto rats, 33% of which died of this disease around day 35 postimmunization. The renal pathology demonstrated widespread glomerular damage and a mononuclear cell infiltration within the interstitial tissue. T cells from immunized rats responded not only to rCol4alpha3NC1, but also to isolated rat GBM. Sera Abs to rCol4alpha3NC1 were detectable in 100% of the rats, but only 20% of the rats had low levels of Ab to isolated rat GBM by Western blot, and none by immunofluorescence. Furthermore, IgG/M binding to or C3 deposition on endogenous GBM in immunized rats were not detected in most of the experimental rats, and showed no statistical correlation with disease severity. Additionally, no electronic dense deposition in the glomeruli was detected in all rats. Those data revealed a disassociation between the disease and anti-GBM Ab. T cell-mediated mechanisms, which are currently under our investigation, may be responsible for the glomerular disease.