IgG auto- and polyreactivities of normal human sera.

Using a panel of self antigens, IgM autoreactivities were clearly and constantly detected by enzyme immunoassay (EIA) in the sera of 29 normal human individuals. Similarly, IgM autoreactivities in sera were reproducibly detected by immunoblotting, using human organ extracts as the antigen sources. In contrast, IgG reactivities were low in whole sera but were considerably increased after affinity-chromatography purification on protein G-Sepharose. These increases differed from one individual IgG preparation to another and from one antigen to another (from 1-94 times) resulting in a unique IgG autoreactivity pattern for each subject. IgG reactivities diminished markedly when the IgG-depleted serum was added to the isolated autologous IgG. IgM antibodies isolated from sera on F(ab')2 IgG immunoadsorbent partially inhibited the binding of IgG to tubulin and myosin but not to actin. The individual IgG preparations examined separately exhibited, with all the autoantigens of the panel, higher autoreactivities than those of the same-but-pooled IgGs, which in turn were higher than those of a commercially available human IgG preparation obtained from approximately 8,000 healthy donors and used for intravenous injection. Depending upon the individual IgG sample, 31-65% of the IgG were bound to a DNP-Sepharose column and were eluted with DNP-glycine. The isolated anti-DNP antibodies were found to be polyreactive and possess higher autoreactivities than the original IgG preparation for all the antigens of the panel. Similarly, IgG antibodies analysed using an antibody exchange procedure were found to be essentially polyreactive but some apparently monospecific antibodies were also noted. These results suggest that the great majority of IgG present in normal humans are composed of polyreactive autoantibodies. IgG autoreactivities are only marginally expressed in these whole sera because of IgM-IgG, IgG-IgG and other, still unidentified, interactions.

Anti-tubulin antibodies in rabbits before and after immunization with pig tubulin.

Sera from rabbits before and after repeated injections of pig tubulin in complete Freund's adjuvant were examined for antibody activity against pig and rabbit tubulins and against a panel of antigens: actin, myosin, DNA, TNP/BSA. Antibody activity against all the antigens of the panel (PAg) increased moderately after the first but not after subsequent injections. Antibody activity against pig and rabbit tubulins strongly increased after the second immunization when the maximum was reached. Isolation of anti-tubulin antibodies from normal or immune sera on tubulin-immunoadsorbent demonstrated the presence of three different antibody populations: (1) polyspecific IgM reacting with the PAg and the tubulins, present in substantial amounts in normal sera and moderately increased in immune sera; (2) small amounts of polyspecific IgG detected only in immune sera; (3) high amounts of specific IgG reacting with pig and rabbit tubulins, present in immune but not normal sera. Western blot analysis of the specific IgG population showed that it contained antibodies reacting with both native pig and rabbit tubulins, as well as antibodies recognizing only the 30,000 proteolytic fragment of pig, but not that of rabbit tubulin. The results indicate that the immunization of rabbits with heterologous tubulin induced specific IgG anti-tubulin antibodies which recognize the self and non-self antigens differently.

Characteristics of Epstein-Barr virus transformed B cell lines from patients with Alzheimer's disease and age-matched controls.

The characteristics of B cell lines isolated from patients with Alzheimer's disease (AD) and age-matched controls were investigated after having been transformed by Epstein-Barr virus (EBV). After isolation of mononuclear blood cells and in vivo or in vitro EBV infection, 35 and 21 lymphoblastoid cell lines (LCLs) were generated from 19 patients with AD (mean age 79.4 years) and 21 age-matched controls (mean age 80.0 years), respectively. B lymphocytes from AD patients were immortalised more easily than those from controls; the percentage of in vitro EBV infected LCLs (B95-LCLs) obtained in the AD group was significantly higher (76.2% versus 33.3% in the control group) and the mean time required for establishment was significantly lower (20.2 and 21.9 days versus 26.7 and 60.9 days in the control group). The EBV receptor and surface immunoglobulin (Ig) analyses showed no difference between the two groups. The expression of Epstein-Barr early antigens (EA) and viral capsid antigens (VCAs) revealed a tendency to higher viral replication in LCLs from AD patients; however, VCA expression remained limited to a small number of cells and did not affect overall cell growth. Finally, qualitative and quantitative differences were observed in the pattern of Ig production. Whereas spontaneously established LCLs from AD patients were generally monoclonal (80% of LCLs versus 33% in the control group), B95-LCLs were all polyclonal and secreted more IgM and IgA than those from controls; the mean IgM level was significantly higher in B95-LCLs from the AD group. These results suggest that B cells derived from AD patients seemed to be less differentiated than cells from age-matched controls.

Detection of tubulin and actin in various cell lines by an immunoperoxidase technique.

This paper reports on the preparation of immunsera against tubulin and actin, and the purification of anti-tubulin and anti-actin antibodies on immunoadsorbent columns. These purified antibodies were used in an indirect immunoperoxidase assay to visualize microtubules and microfilaments in various cell lines. The specificity of antibodies and the methods of cell fixation required are discussed, as well as some aspects of microtubule and microfilament organization, as visualized by this technique.

Bulimia nervosa and autoimmunity.

The autoantibodies that react with dopamine and serotonin are of interest in the study of bulimia nervosa. These neurotransmitters play an important role in appetite control, sexual and social behavior, and stress responses, all of which form a part of the clinical picture of bulimia nervosa. Are these autoantibodies involved in the serotoninergic hypofunctioning present in bulimia nervosa? Are they a part of an immunity regulation system essential for the cerebral system's homeostasis? To address these questions, 31 bulimic females (diagnosed according to DSM-III-R criteria) were compared with 10 control subjects (matched to the patients for sex, age, and demographic/psychosocial features). Measurement of the activity of natural autoantibodies reacting with dopamine, dopamine-beta-hydroxylase and serotonin was performed by an enzyme-linked immunosorbent assay (ELISA) for typical immunoglobulins (IgG, IgM, IgA). All of the autoantibodies of the IgG type were lower in the bulimic group than in the control group, a difference that was statistically significant for IgG anti-serotonin and IgG anti-dopamine. There was a trend for the amount of IgM anti-dopamine to be lower in patients than in controls. Dopamine and serotonin are specific components of brain cells. It can therefore be hypothesized that these antigens acting with autoantibodies could be the antigenic cerebral targets reacting with 'anti-brain' antibodies. The study of these specific autoantibodies provides information about the immunological characteristics that may be related to brain disturbances.

[Bulimia and autoimmunity]. / Boulimie et auto-immunité.

In the first part of this study, we investigated the rate of natural autoantibodies, in a sample of 31 female inpatients with bulimia nervosa according to DSM III-R criteria. The control (age and sex matched) group consisted in high school students including 10 females without eating disorders, depressive disorder or immunological disease. We investigated especially natural autoantibodies reacting with compounds of the central nervous system (Dopamine, Dopamine beta Hydroxylase, Serotonin). Our first conclusion is that there is a lower level of these natural auto-antibodies among female patients with bulimia nervosa. In the second part of the study, we have especially investigated the correlation between impulsivity in bulimia nervosa and the rate of natural autoantibodies against serotonin.

Suppression of anti-DNA antibody production in MRL mice by treatment with anti-idiotypic antibodies.

Antibodies against idiotypic determinants carried by a monoclonal polyspecific natural autoantibody were raised in rabbits and in syngeneic BALB/c mice. These anti-idiotypic antibodies were administered to newborn and to pregnant BALB/c mice and to MRL-lpr/lpr mice. Serial measurements of the idiotypes, naturally occurring autoantibodies, and antibodies obtained after antigenic stimulation were performed in the sera of the injected mice and in the offspring of pregnant mice. No idiotypic suppression was noted in newborn injected mice. Transient suppression of idiotypes recognized by the syngeneic anti-idiotypic antibody was noted in the offspring of pregnant mice injected with the rabbit polyclonal anti-idiotypic antiserum. No changes in naturally occurring autoantibodies or in antibodies appearing after antigenic stimulation were noted in BALB/c mice. In contrast, a significant decrease of spontaneously occurring anti-DNA antibodies was found in MRL-lpr/lpr mice treated with rabbit polyclonal anti-idiotypic antiserum. Furthermore in these mice a slight decrease of anti-TNP antibodies was also observed. These results suggest that anti-idiotypic antibodies directed against natural autoantibodies may play a regulatory role in the immune system; this role is more easily appreciated in mice suffering from immune dysregulation.

Regulation of the humoral immune response by polyspecific natural autoantibodies.

Two different BALB/c IgMk polyspecific monoclonal natural autoantibodies E7 and D23 were administered to neonatal BALB/c mice. When adults, these mice were immunized and challenged with calf myosin, BALB/c actin, human transferrin, calf thymus DNA or TNP-coupled bovine serum albumin (TNP/BSA), in complete Freund's adjuvant. The levels of serum antibody were evaluated by enzyme immunoassay. No differences in anti-actin, anti-transferrin and anti-DNA antibody titres were noted between control and antibody-treated mice. However, anti-myosin antibody titres significantly increased in mice treated with either the E7 or D23 antibody, and anti-TNP antibody titres significantly decreased in mice treated with E7 but not with D23. These differences persisted after antigenic challenge and involved only the IgG response of treated mice. These results suggest that polyspecific natural autoantibodies may be involved in the regulation of the humoral immune response.

Natural antibodies against tubulin, actin myoglobin, thyroglobulin, fetuin, albumin and transferrin are present in normal human sera, and monoclonal immunoglobulins from multiple myeloma and Waldenström's macroglobulinemia may express similar antibody specificities.

Sera from a pool of 800 healthy donors and from 3 individual healthy donors were passed through tubulin, actin, thyroglobulin, myoglobin, fetuin, transferrin and albumin immunoadsorbents. Proteins were eluted in all the immunosorbents and were found to be essentially composed of the three major Ig classes and albumin. The isolated Ig fractions were shown to react specifically, via their Fab fragment with the antigens and were specifically inhibited by them. These results strongly suggest that natural antibodies against the seven antigens are present in normal human serum, and probably against a high variety of self antigens. These results prompted us to search in the sera of patients with monoclonal gammapathies, paraproteins having natural antibody-like function. Among the 62 sera studied 3 were shown to react with actin and 1 with tubulin. Most important, these 4 monoclonal immunoglobulins exhibited similar specificities to that found with natural antibodies. This seems to indicate, that at least for some patients, the monoclonal immunoglobulins produced may represent the expansion of a clone producing a natural antibody.

Studies on active immunization with self antigens. I. Production of antibody to unmodified proteins by neonatal immunization.

Newborn BALB/c mice were repeatedly injected either with syngeneic (BALB/c) or xenogeneic (bovine) myosin, albumin, or actin in sterile physiological saline. The serum antibody response was evaluated by enzyme immunoassay 1 and 2 months after birth and after two booster injections. At 1 month, higher antibody titres were found in the sera of mice injected with syngeneic than with xenogeneic antigens. At 2 months and after boosting, anti-syngeneic actin antibodies were present in equal or higher amounts, anti-syngeneic albumin antibodies were not detected, and anti-syngeneic myosin antibodies were considerably decreased. Antibodies produced after booster injections of syngeneic actin were found to be highly specific and to belong mainly to the IgG isotype. These results suggest that newborn mice are better able than adult mice to respond to stimulation with self antigens, and that administration of self proteins during neonatal life may lead to the induction of immunological memory. They also indicate that one of the primary functions of the immune system in newborn mice is the recognition of self antigens.

Anti-Ia treatment modulates specific and polyclonal antibody responses in Trypanosoma cruzi-infected mice.

Experimental Chagas' disease--infection of mice with the protozoan parasite Trypanosoma cruzi--has been shown to increase the number of Ia-bearing cells in the spleen and the lymph nodes. The majority of these Ia-positive cells were Ig+ and included in the large cell fraction of lymphoid organs from T. cruzi-infected animals indicating that they were activated B cells. These data are consistent with the polyclonal B-cell activation occurring during acute and chronic T. cruzi infection. The levels of secreted natural antibodies, of both IgM and IgG isotypes, were significantly increased in the sera of the infected animals. The present communication demonstrates that in vivo anti-Ia treatment of C3H/HeJ mice infected with the CL strain of T. cruzi suppressed the polyclonal B-cell activation, affecting all the isotypes studied, including IgM, IgG2a and IgG2b, whose levels are predominantly increased during T. cruzi infection. In contrast to the decreased secretion of IgG autoantibodies, the levels of IgM autoantibodies were much less affected. The anti-Ia treatment totally abolished the specific anti-parasite response despite the fact that a pool of Ia-Ig positive cells remained after treatment.

The Epstein-Barr virus-induced production of IgE by human B cells.

B cells, isolated from the blood of healthy individuals and patients allergic to pollen, produced IgE when exposed to the human B-cell polyclonal activator, Epstein-Barr virus (EBV) in vitro and placed in culture. Secreted IgM and IgE were measured using immunoenzymatic assays. No difference was seen between healthy donors and allergic patients in the amount of IgE (or IgM) secreted. Cells were placed in limiting dilution cultures in order to determine the frequency of cells producing IgE or IgM (total and pollen specific) on exposure to EBV. Again, no significant differences in EBV-driven, B-cell precursor frequencies (PF) were seen between normal and allergic individuals. EBV-driven B-cell PF for total IgM and IgE, and pollen-specific IgM and IgE secretion, were 1/450, 1/6500, 1/83,000, and less than 1 per 2,500,000, respectively, for cells from healthy donors, and 1/140, 1/4000, 1/56,000 and less than or equal to 1 per 2,000,000, respectively, for cells from allergic patients. We propose that the increased IgE levels seen in atopic individuals result solely from regulatory defects, rather than an increase in the frequency of B cells committed to the secretion of IgE.

[Anti-actin and anti-tubulin antibodies in the serum of non-immunized animals]. / Anticorps anti-actine et anti-tubuline: leur présence dans les sérums d'animaux non immunisés

The preparation and the purification of anti-action and anti-tubulin antibodies is reported. The occurrence of low amount of IgG anti-tubulin was also found in the sera of non-immunized animals from various species. The staining of the cellular network of microtubules as well as of tubulin paracrystals was observed with both induced and natural antibodies.

Antibodies to tubulin in normal nonimmunized animals.

Sera of normal nonimmunized rabbits, pigs, calves, and humans contain tubulin-reactive antibodies. Usually, low amounts of antibodies against tubulin of the IgG class (2.5-4 mg/100 ml of serum from nonimmunized animals) were isolated. Anti-tubulin antibodies were also produced by injecting pig tubulin in complete Freund's adjuvant into rabbits. Slightly higher amounts of anti-tubulin antibody were isolated from sera of immunized rabbits (7 mg/100 ml of serum). The cytoplasmic network of microtubules of Tcc 36 mouse cells in culture was not clearly stained by natural anti-tubulin antibodies, but dense staining of the centrosphere was observed. In contrast, induced anti-tubulin antibodies densely stained cytoplasmic microtubular networks. Vinblastine-induced tubulin paracrystals were equally stained by natural and induced anti-tubulin antibodies.