Spontaneous autoimmune gastritis and hypochlorhydria are manifest in the ileitis-prone SAMP1/YitFcs mice.

SAMP1/YitFcs mice serve as a model of Crohn's disease, and we have used them to assess gastritis. Gastritis was compared in SAMP1/YitFcs, AKR, and C57BL/6 mice by histology, immunohistochemistry, and flow cytometry. Gastric acid secretion was measured in ligated stomachs, while anti-parietal cell antibodies were assayed by immunofluorescence and enzyme-linked immunosorbent spot assay. SAMP1/YitFcs mice display a corpus-dominant, chronic gastritis with multifocal aggregates of mononuclear cells consisting of T and B lymphocytes. Relatively few aggregates were observed elsewhere in the stomach. The infiltrates in the oxyntic mucosa were associated with the loss of parietal cell mass. AKR mice, the founder strain of the SAMP1/YitFcs, also have gastritis, although they do not develop ileitis. Genetic studies using SAMP1/YitFcs-C57BL/6 congenic mice showed that the genetic regions regulating ileitis had comparable effects on gastritis. The majority of the cells in the aggregates expressed the T cell marker CD3 or the B cell marker B220. Adoptive transfer of SAMP1/YitFcs CD4(+) T helper cells, with or without B cells, into immunodeficient recipients induced a pangastritis and duodenitis. SAMP1/YitFcs and AKR mice manifest hypochlorhydria and anti-parietal cell antibodies. These data suggest that common genetic factors controlling gastroenteric disease in SAMP1/YitFcs mice regulate distinct pathogenic mechanisms causing inflammation in separate sites within the digestive tract.

Aod2, the locus controlling development of atrophy in neonatal thymectomy-induced autoimmune ovarian dysgenesis, co-localizes with Il2, Fgfb, and Idd3.

In genetically susceptible strains of mice, such as A/J and (C57BL/6J x A/J)F1 hybrids, neonatal thymectomy-induced autoimmune ovarian dysgenesis (AOD) is characterized by the development of antiovarian autoantibodies, oophoritis, and atrophy. Temporally, atrophy may be observed during and after the regression of inflammatory infiltrates from the ovary. Histologically, lesions appear as areas devoid of ovarian follicles in all stages of development that have been replaced by luteinized interstitial cells. We report here the mapping of Aod2, the locus that controls this phenotype, to mouse chromosomes 3 within a region encoding Il2 and Fgfb. Most significant, however, is the co-localization of Aod2 to Idd3, a susceptibility gene that plays a role in autoimmune insulin-dependent type 1 diabetes mellitus in the nonobese diabetic mouse.

The relative contribution of the CD28 and gp39 costimulatory pathways in the clonal expansion and pathogenic acquisition of self-reactive T cells.

The zona pellucida (ZP), an ovarian extracellular structure, contains three major glycoproteins: ZP1, ZP2, and ZP3. A ZP3 peptide contains both an autoimmune oophoritis-inducing T cell epitope and a B cell epitope that induces autoantibody to ZP. This study investigates two major T cell costimulation pathways in this disease model. Herein we show that blockage of glycoprotein (gp)39 and CD40 interaction with gp39 monoclonal antibody (mAb) results in the failure to induce both autoimmune oophoritis and autoantibody production. Inhibition of ligand binding to the CD28 receptor with the fusion protein, murine CTLA4-immunoglobulin (Ig), also results in failure to generate antibody to ZP and significantly reduces disease severity and prevalence. Surprisingly, the frequencies of antigen-specific T cells in anti-gp39 mAb-treated mice, CTLA4-Ig treated mice, and in mice given control hamster IgG or control fusion protein L6, were equivalent as determined by limiting dilution analysis (approximately equals 1:5,000). These T cells, which produced comparable amounts of interleukin 4 and interferon gamma in vitro, were able to transfer oophoritis to normal recipients. When anti-gp39 mAb and CTLA4-Ig were given together, the effect was additive, leading to inhibition of T cell activation as determined by in vitro proliferation and limiting dilution analysis (approximately equals 1:190,000); disease and antibody responses were absent in these mice. By studying these two costimulatory pathways in parallel, we have shown that autoimmune disease and autoantibody production are inhibitable by blocking either the gp39 or the CD28 pathway, whereas inhibition of clonal expansion of the effector T cell population occurs only when both pathways are blocked.

The black mink (Mustela vison). A natural model of immunologic male infertility.

Breeding for fine black fur has generated a colony of mink wherein 20-30% of the males are infertile. Two clinical groups are distinguishable: one being infertile from the start (primary infertility), and the other infertile after one or more years of fertility (secondary fertility). Although the etiology of primary infertility is unknown, the available data indicate that secondary infertility is associated with an autoimmune disease of the testis. Thus, male mink with secondary infertility have (a) higher prevalence and levels of anti-sperm antibody when compared with animals with primary infertility, and the antibody prevalence varies with fur color; (b) severe monocytic orchitis (47%) and/or aspermatogenesis (75%) with negative cultures for bacterial, fungal, mumps, or Coxsackie B viral organisms; (c) massive and extensive granular deposits of mink IgG and/or C3 (71%), typical of immune complexes, along the basal lamina of seminiferous tubules; (d) testes that when eluted with buffer or low pH yielded IgG that was 10-fold enriched in anti-sperm antibody activity as compared with serum IgG; and (e) no immunopathologic evidence of Aleutian mink disease. Although the sperm antigen-antibody complexes in the testis may be important as a pathogenetic mechanism of the testicular disease, there is no correlation between fluorescent anti-sperm antibody detection in the serum and the infertile state. The infertile black mink is a new model of infertility associated with naturally occurring autoimmune disease of the testis.

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.

Allergic orchitis lesions are adoptively transferred from vasoligated guinea pigs to syngeneic recipients.

Histopathology typical of allergic orchitis developed in testes of inbred guinea pigs 16 months after vasoligation. A similar histopathology was found in unoperated testes after unilateral vasoligation. Peritoneal exudate cells from vasoligated guinea pigs transferred identical lesions to syngeneic recipients. The testicular lesions in long-term vasoligated guinea pigs have an immunological basis.

Autoantibodies from vasectomized guinea pigs inhibit fertilization in vitro.

Immunoglobulin G and Fab antibodies were isolated from the serum of vasectomized guinea pigs, and the effects of the antibodies on fertilization in vitro were investigated. These antibodies had profound inhibitory effects on (i) sperm-to-sperm adhesion, (ii) the acrosome reaction, (iii) sperm-zona binding, and (iv) sperm-ovum fusion. This finding may explain certain cases of infertility after vasovasostomy in men.

Neonatal thymectomy results in a repertoire enriched in T cells deleted in adult thymus.

In B6AF1 mice, T lymphocytes that use the V beta 11-positive (and not V beta 6-positive or V beta 8-positive) segment in their receptor for antigen are greatly reduced in the thymus and peripheral lymphoid tissues, most likely as a result of clonal deletion. The relative number of V beta 11-positive cells in adult lymph nodes was ten times as high in B6AF1 mice thymectomized 1 to 4 days after birth as in normal mice. Moreover, for the first 10 days of life of B6AF1 mice, mature V beta 11-positive T cells were readily detected in the thymus and spleen. Thus neonatal thymectomy results in the maintenance of the receptor repertoire of early postnatal life, and this correlates with the subsequent development of organ-specific autoimmune diseases.

Bax-deficient mice with lymphoid hyperplasia and male germ cell death.

BAX, a heterodimeric partner of BCL2, counters BCL2 and promotes apoptosis in gain-of-function experiments. A Bax knockout mouse was generated that proved viable but displayed lineage-specific aberrations in cell death. Thymocytes and B cells in this mouse displayed hyperplasia, and Bax-deficient ovaries contained unusual atretic follicles with excess granulosa cells. In contrast, Bax-deficient males were infertile as a result of disordered seminiferous tubules with an accumulation of atypical premeiotic germ cells, but no mature haploid sperm. Multinucleated giant cells and dysplastic cells accompanied massive cell death. Thus, the loss of Bax results in hyperplasia or hypoplasia, depending on the cellular context.

Antigen mimicry in autoimmune disease sharing of amino acid residues critical for pathogenic T cell activation.

A nonamer peptide from murine nicotinic acetylcholine receptor delta chain (ACR delta), which shared four amino acid residues with a nonamer peptide of murine ovarian zona pellucida glycoprotein ZP3, induced murine autoimmune oophoritis and IgG autoantibody to the zona pellucida. Crossreaction between the ACR delta and ZP3 peptides was established by the response of a ZP3 peptide-specific, oophoritogenic T cell clone to both peptides in association with IA (alpha k beta b). By substituting the ZP3 peptides with a single alanine, four amino acids within the ZP3 peptide were found to be important for ovarian autoimmune disease, autoantibody response, and stimulation of the ZP3-specific T cell clone. Substitution with conservative amino acids of three residues also ablated activity, whereas the fourth, a phenylalanine, was replaceable by tyrosine without loss of activity. Of the four critical amino acids, three were shared between the ZP3 peptide and the ACR delta peptide. Moreover, polyalanine peptides with the four critical ZP3 amino acids or the four amino acids common to the ZP3 and ACR delta peptides induced immune response to ZP3 and elicited severe ovarian autoimmune disease. Thus, organ-specific autoimmune disease can occur through immune response against unrelated self (or foreign) peptides that share with a self-peptide sufficient common amino acid residues critical for activation of pathogenic, autoreactive T cells.

Application of the solid phase C1q and Raji cell radioimmune assays for the detection of circulating immune complexes in glomerulonephritis.

The C1q solid phase and Raji cell radioimmune assays were used to determine the frequency of detectable circulating immune complexes in patients with glomerulonephritis. In this study, 46% of 56 patients with glomerulonephritis had evidence of circulating immune complexes. More important, circulating immune complexes were associated with some, but not other, types of glomerulonephritis. Thus, immune complexes were detected in lupus glomerulonephritis (9/9 patients), rapidly progressive glomerulonephritis (5/6 patients), and acute nephritis (5/6 patients), but not in IgA-IgG glomerulonephritis (0/7 patients), or membranous glomerulonephritis (0/8 patients). The Raji cell radioimmune assay and the C1q solid phase radioimmune assay showed concordance of 79% in the detection of circulating immune complexes. Serial determinations, in general, showed either persistence of a negative or positive result of conversion of positive to negative.

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.

Circulating immune complexes in Lyme arthritis. Detection by the 125I-C1q binding, C1q solid phase, and Raji cell assays.

We have found immunoglobulin (Ig) G-containing material consistent with immune complexes in the sera of patients with Lyme arthritis. It was detected in 29 of 55 sera (55%) from 31 patients by at least one of three assays: (125)I-C1q binding, C1q solid phase, or Raji cell. The presence of reactive material correlated with clinical aspects of disease activity; it was found early in the illness, was most prominent in sera from the sickest patients, was infrequent during remissions, and often fluctuated in parallel with changes in clinical status. The results in the two C1q assays showed a strong positive correlation (P<0.001). They were each elevated in 45% of the sera and were usually concordant (85%). In contrast, the Raji cell assay was less frequently positive and often discordant with the C1q assays. In sucrose density gradients, putative circulating immune complexes sedimented near 19S; they, too, were detected best by the two assays based on C1q binding. An additional 7S component was found in some sera by the (125)I-C1q binding assay. Serum complement was often above the range of normal in patients with mild disease and normal in patients with severe disease but did not correlate significantly with levels of circulating immune complexes. IgM and IgG rheumatoid factors were not detectable. These findings support a role for immune complexes in the pathogenesis of Lyme arthritis. Their measurement, by either the (125)I-C1q binding assay or by the C1q solid phase assay, often provides a sensitive index of disease activity. Moreover, the complexes are likely sources of disease-related antigens for further study of this new disorder.

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.

SRN1, a yeast gene involved in RNA processing, is identical to HEX2/REG1, a negative regulator in glucose repression.

The yeast RNA1 gene encodes a cytosolic protein that affects pre-tRNA splicing, pre-rRNA processing, the production of mRNA, and the export of RNA from the nucleus to the cytosol. In an attempt to understand how the RNA1 protein affects such a diverse set of processes, we sought second-site suppressors of a mutation, rna1-1, of the RNA1 locus. Mutations in a single complementation group were obtained. These lesions proved to be in the same gene, SRN1, identified previously in a search for second-site suppressors of mutations that affect the removal of intervening sequences from pre-mRNAs. The SRN1 gene was mapped, cloned, and sequenced. DNA sequence analysis and the phenotype of disruption mutations showed that, surprisingly, SRN1 is identical to HEX2/REG1, a gene that negatively regulates glucose-repressible genes. Interestingly, SRN1 is not a negative regulator of RNA1 at the transcriptional, translational, or protein stability level. However, SRN1 does regulate the level of two newly discovered antigens, p43 and p70, one of which is not glucose repressible. These studies for the first time link RNA processing and carbon catabolite repression.

Localization of a highly divergent mammalian testicular alpha tubulin that is not detectable in brain.

Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.

Autoimmune ovarian disease in day 3-thymectomized mice: the neonatal time window, antigen specificity of disease suppression, and genetic control.

Discovery of the CD4+CD25+ T cells has stemmed from investigation of the AOD in the d3tx mice. Besides CD4+CD25+ T cell depletion, d3tx disease induction requires effector T cell activation prompted by lymphopenia. This is supported by other neonatal AOD models in which T cell-mediated injury has been found to be triggered by immune complex or Ag immunization. In addition, there is growing evidence that support a state of neonatal propensity to autoimmunity, which depends on concomitant endogenous antigenic stimulation, concomitant nematode infection, resistance to CD4+CD25+ T cell regulation, and participation of the neonatal innate system. The suppression of d3tx disease by polyclonal CD4+CD25+ T cells appears to be dependent on endogenous Ag and the persistence of regulatory T cells. Thus, suppression of AOD occurs in the ovarian LN, and AOD emerges upon ablation of the input regulatory T cells; and in AIP, the hormone-induced expression of prostate Ag in the CD4+CD25+ T cell donors rapidly enhances the capacity to suppress disease over Ag negative donors. Finally, genetic analysis of AOD and its component phenotypes has uncovered seven Aod loci. As the general themes that emerged, significant epistatic interactions among the loci play a role in controlling disease susceptibility, the majority of the Aod loci are linked to susceptibility loci of other autoimmune diseases, and the genetic intervals encompass candidate genes that are differentially expressed between CD4+CD25+ T cells and other T cells. The candidate genes include Pdcd1, TNFR superfamily genes, H2, Il2, Tgfb, Nalp5 or Mater, an oocyte autoAg that reacts with autoantibody in sera of d3tx mice.

T-lymphocyte subpopulations in peripheral blood and tissues of cancer patients.

Patients with cancer often show impaired immune functions; however, the basis of this suppression is still not understood. In several experimental systems, human T-cells with receptors for Fc of immunoglobulin G may function as suppressors, and those with receptors for Fc of immunoglobulin M may function as helpers. Peripheral blood as well as tumor tissue infiltrates were examined for proportions and numbers of T gamma, T mu, or Ia-positive T-cells. Forty-five untreated patients with solid tumors and 24 patients with lymphomas were studied. An increase in the percentage of peripheral blood T gamma cells (p < 0.001) and a decrease in T mu cells (p < 0.0005) were recorded in all tumor patients when compared with 30 normal controls. Percentages and absolute numbers of peripheral blood Ia-positive T-cells were decreased (p < 0.001 and < 0.00001) in solid-tumor patients; by contrast, the proportion of peripheral blood Ia-positive T-cells was elevated (p < 0.005) in lymphoma subjects. Studies of cancer tissues from 46 untreated patients using immunofluorescence and mouse hybridoma antibody specific for T-cells showed that tumor lymphocytic infiltrates were composed mainly of T-cells. Double staining with fluorescein-conjugated specific anti-T gamma and Ia-positive T-cells within solid-tumor lymphoid infiltrates. A comparison of peripheral blood and tumor lymphocyte T-cell profiles revealed that, in some patients, low proportions of Ia-positive T-cells in blood were paralleled by a high percentage of such cells in tumor lymphoid infiltrates.

Meiotic chromosome morphology and behavior in zip1 mutants of Saccharomyces cerevisiae.

The yeast Zip1 protein (Zip1p) is a component of the central region of the synaptonemal complex (SC). Zip1p is predicted to form a dimer consisting of a coiled-coil domain flanked by globular domains. To analyze the organization of Zip1p within the SC, in-frame deletions of ZIP1 were constructed and analyzed. The results demonstrate that the C terminus but not the N terminus of Zip1p is required for its localization to chromosomes. Deletions in the carboxy half of the predicted coiled-coil region cause decreases in the width of the SC. Based on these results, a model for the organization of Zip1p within the SC is proposed. zip1 deletion mutations were also examined for their effects on sporulation, spore viability, crossing over, and crossover interference. The results demonstrate that the extent of synapsis is positively correlated with the levels of spore viability, crossing over, and crossover interference. In contrast, the role of Zip1p in synapsis is separable from its role in meiotic cell cycle progression. zip1 mutants display interval-specific effects on crossing over.

Histoplasmosis. Association with circulating immune complexes, eosinophilia, and mesangiopathic glomerulonephritis.

A patient with disseminated histoplasmosis, eosinophilia, and transient mesangiopathic glomerulonephritis stimulated a search for the presence of circulating immune complexes. Serum samples obtained on the fifth and 11th hospital days were strongly positive for ciculating immune complexes by both the Raji cell radioassay and the C1q solid phase assay. During the course of complete clinical recovery without therapy, both assays were weakly positive for circulating immune complexes on day 33. On day 56 they were negative. Using this case as a prototype, possible mechanisms for the renal immunopathology and the eosinophilic response are discussed with reference to the immunological perturbations thay may be observed in systemic mycotic infection.