Cardiac myosin induces myocarditis in genetically predisposed mice

After infection with coxsackie virus B3 (CB3), H-2 congenic mice on an A- background develop immunologically mediated myocarditis associated with an increased titer of myosin autoantibody, part of which is specific for the cardiac myosin isoform. The present study demonstrates that cardiac myosin itself induces severe myocarditis and high titers of myosin autoantibodies in A/J, A.SW/SnJ, and A.CA/SnJ mice. As in CB3-induced myocarditis, one population of these autoantibodies was specific for cardiac myosin. A.BY/SnJ and B10.A/SgSnJ mice also developed the disease after immunization, but the prevalence and the myosin autoantibody titers were lower. In contrast, C57BL/6J and C57BL/10J mice were resistant to myocarditis induced by cardiac myosin and did not develop increased myosin autoantibodies or cardiac myosin-specific autoantibodies. Immunization with skeletal muscle myosin had no effect compared with controls injected with complete Freund's adjuvant, thereby suggesting that the immunogenic epitopes are unique to the cardiac myosin isoform. Furthermore, we found that susceptibility to myocarditis induced by cardiac myosin is influenced by the major histocompatibility complex and by genes not closely linked to the major histocompatibility complex. Because there are parallels between myocarditis induced by cardiac myosin and that induced by CB3, this new animal model can be used to analyze the pathologic mechanisms in autoimmune heart disease.     

Inhibition of H-Y cell-mediated cytolysis by monoclonal H-Y-specific antibody

Assays of H-Y-specific, cell-mediated cytolysis (CMC) in vitro were carried out with B6 female effector cells and B6 male target cells. Monoclonal H-Y antibody was added to the lytic assay to test whether the antigenic determinant(s) involved in H-Y-specific CMC was distinct from the serologically detected H-Y antigen. Significant blocking was observed, suggesting that the H-Y antigen detectable serologically is similar to H-Y antigen recognized by cytotoxic T cells.Abbreviations used in this paper B6 C57BL/6 - BALB BALB/c - CMC cell-mediated cytolysis - E effector cells - T target cells     

In situ nick translation of metaphase chromosomes with biotin-labeled d-UTP

Summary Conditioned culture medium from Daudi cells was used as a source of soluble H-Y antigen. Concentrated culture medium was labeled with ¹²⁵I and then fractionated by gel filtration. Column fractions were assayed for the presence of H-Y antigen by urease-ELISA. H-Y antigen-containing fractions were then pooled and subjected to an improved immunoprecipitation protocol. Three predominant H-Y antigenic proteins were identified with estimated molecular weights of above 200,000, 50,000, and 20,000.     

Variations in the susceptibility to Coxsackievirus B3-induced myocarditis among different strains of mice

This study was undertaken to examine the inherent predisposition of different inbred strains of mice to develop Coxsackievirus B3-induced myocarditis. A time course study established the pertinent, differential parameters of the disease and their corresponding genetic control. The A.BY/SnJ (H-2b), A.SW/SnJ (H-2s), A.CA/SnJ (H-2f), B10.S/SgSf (H-2s), B10.PL/SgSf (H-2u), and C3H.NB/SnJ (H-2p) strains were found to vary widely in the extent and duration of viremia, in the temporal appearance and titer of neutralizing antibody, and in the prevalence, severity, and duration of myocardial disease. The A.BY/SnJ (H-2b), A.SW/SnJ (H-2s), A.CA/SnJ (H-2f), and C3H.NB/SnJ (H-2p) mice developed continuing, chronic myocardial disease, whereas B10.S/SgSf (H-2s) and B10.PL/SgSf (H-2u) did not. The four strains that displayed prolonged myocarditis also produced heart-specific myocardial autoantibodies. Heart-specific autoantibodies were not found in the B10.S/SgSf and B10.PL/SgSf animals. Differences in prevalence and titer of these heart-specific autoantibodies were noted among the three A strain H-2 congenic lines. The influence of the major histocompatibility complex (MHC) on disease production was demonstrated by comparison of the three A strain and two B10 strain H-2 congenics. Differences between A.SW/SnJ (H-2s) and B10.S/SgSf (H-2s) suggested non-MHC control of disease. These studies additionally indicate that the genetic regulation of susceptibility to CB3 infection and the direct virus-induced inflammation differ from the later immunopathic myocarditis.     

H-Y transplantation antigen in human XO females

Summary When sensitized with human cultured fibroblasts of the XY and XO, but not XX, sex chromosomal types C57BL/6 female mice reject syngeneic male grafts accelerated (second set graft reaction). These findings demonstrate that the antigenic determinants of H-Y antigen of man and mouse are homologous and that XO females (at least those tested) carry the H-Y transplantation antigen. The results are discussed in the light of the question of differences between the H-Y antigen as defined by grafting and serology and the chromosomal localization of the H-Y structural gene(s).     

The affinity/avidity and length of exposure to the deleting ligand determine dependence on CD28 for the efficient deletion of self-specific CD4+CD8+ thymocytes

Whether the CD28/B7 signaling pathway is essential for the negative selection of immature CD4+CD8+ (DP) thymocytes expressing self-specific alphabeta TCRs is a controversial issue. In this study we examined the role of CD28 in the deletion of thymocytes that express either the H-Y or the 2C transgenic TCR. In H-2(b) male mice that expressed the H-Y TCR, negative selection of DP H-Y TCR+ thymocytes occurred very efficiently and this deletion was unaffected by the CD28(-/-) mutation. In H-2(b) 2C mice, where the deletion of DP 2C TCR+ thymocytes occurred less efficiently, the CD28(-/-) mutation led to a higher recovery of DP thymocytes. Using an in vitro deletion assay, a requirement for the CD28 signaling pathway in the deletion of DP H-Y TCR+ thymocytes was evident at low, but not high, densities of the antigenic ligand. Similar results were also observed in an in vivo assay for the deletion of these thymocytes. Intraperitoneal administration of an anti-CD3epsilon mAb led to the intrathymic deletion of DP H-Y TCR+ thymocytes in a CD28-dependent manner at the 24-h time point. However, the CD28 dependence was less evident at the 40-h time point. These results indicate that the dependence on CD28 for the efficient deletion of self-specific thymocytes is determined by the concentration, affinity/avidity, and length of exposure to the deleting ligand.     

XY female mice express H-Y antigen

Karyotypically XY individuals of the C57BL/6J-Y^POS mouse stock develop as females or hermaphrodites, but never as normal males. The aberrant sexual development results from the interaction of the C57BL/6J genetic background with the M. poschiavinus-derived Y chromosome. XY females from this stock were assayed for H-Y antigen. By the criteria of skin-grafting, the cell-mediated lympholysis test, and the popliteal lymph node assay, these XY females are antigenically indistinguishable from normal C57BL/6 males. Implications for the hypothesis that H-Y antigen induces formation of the mammalian testis are discussed.     

H-Y antigen in transsexuality,and how to explain testis differentiation in H-Y antigen-negative males and ovary differentiation in H-Y antigen-positive females

Summary H-Y antigen was determined in eight transsexual patients. Two of the four male-to-female transsexual patients typed as H-Y antigen-negative, while the other two typed as expected from their phenotypic and gonadal sex, namely H-Y antigen-positive. Of the four female-to-male transsexual patients, three typed as H-Y antigen-positive and one was H-Y antigen-negative, as expected. The presence of normal testes in H-Y antigen-negative males is assumed to result from a mutation of nucleotide sequences of the H-Y structural gene for antigenic determinants. Thus, an H-Y is produced with normal receptor-binding activity which can sustain the testis determination of the bipotent gonadal anlage. In the case of H-Y antigen-positive females with normal ovaries a deletion of the autosomally located H-Y structural gene is assumed. This deletion should affect sequences for repressor-binding (as was suggested for H-Y antigen-positive XX-males) and for receptor-binding activity of the H-Y antigen molecule. The resulting H-Y antigen is unable to bind to the gonadal receptor of the bipotent gonadal anlage. Thus an ovary is determined. The relevance of H-Y antigen for the aetiology of transsexualism is discussed.     

Behavioural studies of MHC‐congenic mice

Behavioural studies of MHC‐congenic mice and rats have focused primarily on mate choice and the ability to discriminate between strains by their urine odours, but these strains may differ in other behaviours, such as activity and ultrasonic vocalizations. Ivanyi (1978, Proc. Roy. Soc. Lord. 202, 117–158) has reviewed the physiological differences associated with the MHC, many of which could influence behaviour. We have started a systematic study of behavioural development and adult behaviour in MHC‐congenic mice. A developmental test battery (growth, rate, locomotion, grooming, eye opening, ultrasonic vocalizations, etc.) was used to examine differences between C57BL/6J vs. B6‐H‐2^bm1 and C57BL/10SnJ vs. B10.BR/sgSnJ mice. A test battery of spontaneous behaviours (activity, exploration, ultrasonic vocalizations, etc.) was used to examine behavioural differences between adult C57BL/6J vs. B6‐H‐2^bm1 and C57BL/10SnJ vs. B10.BR/sgSnJ mice. Differences in development and in adult behaviours between these MHC-congenic strains is discussed in relation to possible neural, endocrine and immune system differences. Future studies will compare MHC-congenic mice on levels of anxiety, sociosexual behaviour and on learning paradigms. This revised version was published online in July 2006 with corrections to the Cover Date.     

H-Y antigen expression in different tissues from transsexuals

Summary H-Y-antigen expression was analyzed in patients with transsexuality. Peripheral blood lymphocytes and various tissues were examined using the cytotoxicity assay of Goldberg et al. (1971). Peripheral blood lymphocytes from healthy male and female subjects were used as controls as well as tissues from nontranssexual individuals and from male and female C57Bl/6J mice. In three female-to-male transsexuals the peripheral blood lymphocytes were H-Y antigen positive. In these patients also their ovaries, uterus, and mammae were found to be H-Y antigen positive. Three male-to-female transsexuals were examined. The peripheral blood lymphocytes in two of these patients were found to be H-Y antigen negative. Their testes were also H-Y antigen negative, as well as the epididymus, the corpus cavernosum penis, and the cremaster muscle which was analyzed in one of them. One male-to-female transsexual had peripheral blood lymphocytes which were H-Y antigen positive; this patient had testis and corpus cavernosum penis which were also H-Y-antigen positive.     

Major genes control hormone-induced ovulation rate in mice

The present study examined the magnitude of genetic variation, mode of inheritance and number of loci controlling major genetic differences in hormone-induced ovulation rate in mice. Mice were injected with 5 i.u. PMSG at 28 days of age and 5 i.u. hCG at 30 days, and hormone-induced ovulation rate was determined from counts of oviducal eggs in cumulus the next morning. Six-fold genetic differences in induced ovulation rate were detected amongst strains, ranging from a low mean (+/- s.e.) value of 8.8 (+/- 0.9) in A/J up to 53.5 (+/- 2.2) in C57BL/6J. The number of ova differed significantly amongst strains and amongst F1 crosses (P less than 0.0001): 70% of the variation in hormone-induced ovulation rate was amongst strains. Of 9 F1 crosses examined, 4 showed positive heterosis, 3 showed no heterosis and 2 showed negative heterosis for hormone-induced ovulation rate. Analysis of parental, F1 and F2 generations revealed that the induced ovulation rate of A/J and C57BL/6J mice differed due to the action of about 3 or 4 loci, and A.SW/SnJ and SJL/J mice differed due to the action of about 2 to 3 loci. Analysis of recombinant inbred strains formed from A/J and C57BL/6J confirmed that these strains differed due to the action of a small number of loci. This study demonstrates the existence of a small number of major genes controlling hormone-induced ovulation rate in young mice.     

H-Y Antigen Expression in Temperature Sex-Reversed Turtles (Emys orbicularis)

H-Y antigen has been used as a marker for the heterogametic sex and is assumed to be an organizing factor for the heterogametic gonad. In the turtle Emys orbicularis , H-Y antigen is restricted to the female cells, indicating a female heterogamety (ZZ/ZW) sex-determining mechanism. Moreover, the sexual differentiation of the gonads is temperature sensitive, and complete sex reversal can be obtained at will. In this framework the relationships between H-Y antigen, temperature, and gonadal phenotype were studied. Mouse H-Y antiserum was absorbed with blood and gonadal cells of control wild male and female adults, and with blood and gonadal cells from three lots of young turtles from eggs incubated at 25–26°C (100% phenotypic males), at 30–30.5°C (100% phenotypic females), or at 28.5–29°C (majority of females with some males and intersexes). The residual activity of H-Y antiserum was then estimated using an immunobacterial rosette technique. In adults, both blood cells and gonadal cells were typed as H-Y negative in males and as H-Y positive in females. In each of the three lots of young, blood cells were H-Y negative in some individuals and H-Y positive in others. The proposed interpretation is that the H-Y negative individuals were genotypic males (ZZ) and the H-Y positive were genotypic females (ZW). The gonads of these animals were then pooled in different sets according to their sexual phenotype and to the presumed genotypic sex (i.e., blood H-Y phenotype). Testicular cells were typed as H-Y negative in genotypic males as well as in the presumed sex-reversed genotypic females; likewise, ovarian cells were typed as H-Y positive in genotypic females as well as in the presumed sex-reversed genotypic males. These results provide additional evidence that H-Y antigen expression is closely associated with ovarian structure in vertebrates displaying a ZZ/ZW sex-determining mechanism.     

Complement activation by cell-associated immune complexes in contact sensitivity

Lymph node cells collected 4 days after painting the skin with picryl chloride activate the first components of the classical pathway of complement cascade, as shown by consumption of C4 of rabbit complement with total sparing of C5 and factor B activity. In contrast, lymph node cells collected 1 or 6 days after sensitization fail to do so. The ability of “4-day” cells to activate complement is inhibited by treating the cells with specific low-molecular-weight hapten, which is known to dissociate the immune complex present on the cell surface. When mouse serum was used as source of complement, a different behavior in complement activation between CBA/J and B.10.D2-New/SnJ serum was observed: “4-day” cells failed to consume CBA/J serum whereas a normal complement activation was detected when B.10.D2-New/SnJ serum was used. Using these two sera which differ in the level of C4, an inverse relationship between the ability of “4-day” cells to activate complement and their capacity to induce contact sensitivity when injected into the footpad of normal recipients was reported. Experiments performed using sera from C5 genetically deficient mice demonstrate that only the early complement components are involved, suggesting that membrane immune complexes are solubilized as a result of complement activation; on the other hand, membrane bound activated complement components could alter the immunizing potential of “4-day” cells.     

Anti-receptor antibody-induced H-Y-specific delayed-type hypersensitivity responses in nonresponder mice

The present study examines an antiserum prepared against antigen-reactive T cells that induces murine H-Y-specific delayed-type hypersensitivity (DTH) responses. This anti-H-Y receptor antibody (ARA) was raised in C57BL/6 male mice against splenic T lymphocytes from H-Y immune syngeneic females. Subcutaneous administration of ARA to cyclophosphamide-pretreated C57BL/6 females is able to induce H-Y-specific delayed-type footpad swelling responses. The DTH inducing capacity in ARA was selectively retained on rabbit anti-mouse immunoglobulin columns and was absorbed completely by H-Y immune lymphoid cells from C57BL/6 females. The induction of H-Y DTH reactivity was due at least in part to the activation of H-Y antigen-specific T lymphocytes that could adoptively transfer DTH-like responses to naive female mice. ARA induces DTH responses in strains with the same lgh regions, including selected strains of H-Y nonresponders. Therefore, MHC-linked lr genes do not appear to be as critical when responses are triggered by ARA instead of by antigen. Possible mechanisms for the induction of immune responses by ARA are discussed.     

Frequency and cross-reactivity od cytolytic T lymphocyte precursors reacting against male alloantigens

It is well established that cytotoxic T lymphocytes (CTL) specific for the male minor histocompatibility antigen (H-Y) are generated by restimulation in vitro of in vivo primed spleen cells from C57BL/6 (H-2b) female mice with syngeneic male spleen cells. When tested on target cells from H-2 different strains, the male-specific C57BL/6 CTL populations exhibited significant lysis of DBA/2 (H-2d), A (H-2a), but not C3H (H-2k), male and female target cells. In an attempt to document this cross-reactivity further at the clonal level, a sensitive technique of limiting dilution analysis was used to determine the specificity of C57BL/6 individual CTL precursors (CTL-P) reactive against the male antigen. The mean frequency of anti-H-Y CTL-P in spleens of primed female mice was about 1/3500. Between one-third to one-tenth of these CTL-P produced a progeny that cross-reacted with H-2d (allogeneic) female target cells. These findings were confirmed by the analysis of the reactivity pattern exhibited by male-specific CTL clones derived by limiting dilution. Of 99 clones tested, 13 were found to cross-react with female DBA/2 target cells. These results thus indicate that a relatively large proportion (greater than 10%) of H-2b CTL-P directed against the H-Y antigen cross-react with target cells expressing H-2d alloantigens in the absence of H-Y antigen.     

Lipopolysaccharide receptors on lymphocytes. I. Lack of immunologic recognition of a putative LPS receptor on LPS-responder lymphocytes by LPS-nonresponder mice

We have examined the potential immunogenicity of viable lymphocytes from C3HeB/FeJ responder mice adoptively transferred into congenic nonirradiated C3H/HeJ nonresponder mice. Immunologic rejection or acceptance of donor cells was employed as indirect evidence for the presence or absence of an antigenically distinct "LPS receptor" present on donor lymphocytes. Immunogenicity was evaluated by in vitro assessment of the subsequent proliferative response of recipient splenocytes to protein-free LPS after multiple i.p. injections of responder lymphocytes. Control experiments have made use of syngeneic donor lymphocytes differing immunologically by the presence of the H-Y minor histocompatibility antigen. The results of these experiments provide evidence for the concept that if the phenotypic difference between LPS responder and nonresponder mice is also expressed antigenically in the form of an LPS receptor, that antigenic difference is significantly less immunogenic than a minor histocompatibility antigen.     

A protocol for high-throughput phenotyping, suitable for quantitative trait analysis in mice

Whole-genome genetic association studies in outbred mouse populations represent a novel approach to identifying the molecular basis of naturally occurring genetic variants, the major source of quantitative variation between inbred strains of mice. Measuring multiple phenotypes in parallel on each mouse would make the approach cost effective, but protocols for phenotyping on a large enough scale have not been developed. In this article we describe the development and deployment of a protocol to collect measures on three models of human disease (anxiety, type II diabetes, and asthma) as well as measures of mouse blood biochemistry, immunology, and hematology. We report that the protocol delivers highly significant differences among the eight inbred strains (A/J, AKR/J, BALBc/J, CBA/J, C3H/HeJ, C57BL/6 J, DBA/2 J, and LP/J), the progenitors of a genetically heterogeneous stock (HS) of mice. We report the successful collection of multiple phenotypes from 2000 outbred HS animals. The phenotypes measured in the protocol form the basis of a large-scale investigation into the genetic basis of complex traits in mice designed to examine interactions between genes and between genes and environment, as well as the main effects of genetic variants on phenotypes.     

血清学检测H-Y噬菌体Fab抗体阳性克隆的分析

目的 为分析H-Y噬菌体Fab抗体特异性,筛选用于抗体亲和力提高的H-Y噬菌体Fab抗体阳性克隆.方法 以从噬菌体Fab抗体库中筛选到具有雄性特异性结合活性的阳性克隆A6、A8、E6为基础,通过C57BL/6鼠脾细胞为抗原的ELISA分析3株阳性克隆的特异性,镜下观察亲和力较好的A8阳性克隆ELISA结果,利用生物信息学方法预测分析该克隆的抗体基因可变区序列和结构.结果 ELISA分析显示3株阳性克隆具有雄性特异性,其中A8阳性克隆具备较好的雄性特异性.A8克隆具有免疫球蛋白轻链和重链可变区结构,其重链、轻链可变区分别属于VHI和VκIV基因家族.结论 A8阳性克隆可用于后续的导向筛选和抗体基因改造等研究工作.