Background: Many and diverse autoimmune abnormalities have been reported in children with autism. Natural autoantibodies (NAAbs) play important immunoregulatory roles in recognition of the immune self. The objective of this study was to examine the presence of NAAbs in the sera of children with autism and across severity subgroups of autistic behavioral impairments. Methods: NAAbs were titrated in sera through an ELISA procedure in 60 low-functioning children with autism and 112 typically developing controls matched for age, sex and puberty. Results: Serum titers of IgG anti-F(ab')(2) autoantibodies were significantly lower in children with autism compared to typically developing controls (p < 0.0001), and were significantly negatively associated with autism severity (p = 0.0001). This data appears to be related more specifically to autism than to intellectual disability, given that IgG anti-F(ab')(2) levels were significantly negatively correlated with IQ scores in the autism group (p = 0.01). Conclusions: This is the first report in autism of abnormally low natural anti-F(ab')(2) autoantibody activity. The findings suggest a dysfunction of self-recognition mechanisms which may play a role in the pathogenesis of autism, especially for the severely affected children. These findings strengthen the hypothesis of an autoimmune process in autism and open the prospect of alternative medical treatment. Further neuroimmunological research is warranted to understand the exact mechanisms underlying this reduced natural IgG anti-F (ab')(2) autoantibody activity, and to assess its impact on the pathophysiology and behavioral expression of autism.

OPINION article Front. Immunol., 12 June 2018Sec. Comparative Immunology Volume 9 - 2018 | https://doi.org/10.3389/fimmu.2018.01320

In this review, we summarize and discuss some key findings from the study of naturally occurring autoantibodies. The B-cell compartment of the immune system appears to recognize almost all endogenous and environmental antigens. This ability is accomplished principally through autopolyreactive humoral and cellular immune receptors. This extended autopolyreactivity (1) along immunoglobulin gene recombination contributes to the immune system's ability to recognize a very large number of self and non-self constituents; and (2) generates a vast immune network that creates communication channels between the organism's interior and exterior. Thus, the immune system continuously evolves depending on the internal and external stimuli it encounters. Furthermore, this far-reaching network's existence implies activities resembling those of classical biological factors or activities that modulate the function of other classical biological factors. A few such antibodies have already been found. Another important concept is that natural autoantibodies are highly dependent on the presence or absence of commensal microbes in the organism. These results are in line with past and recent findings showing the fundamental influence of the microbiota on proper immune system development, and necessitate the existence of a host-microbe homeostasis. This homeostasis requires that the participating humoral and cellular receptors are able to recognize self-antigens and commensal microbes without damaging them. Autopolyreactive immune receptors expressing low affinity for both types of antigens fulfil this role. The immune system appears to play a holistic role similar to that of the nervous system.

SummaryWe have generated three monoclonal cell‐penetrating antibodies (CPAbs) from a non‐immunized lupus‐prone (NZB × NZW)F1 mouse that exhibited high anti‐DNA serum titres. These CPAbs are polyreactive because they bind to DNA and other cellular components, and localize mainly in the nucleus of HeLa cells, albeit with a distinct nuclear labelling profile. Herein, we have examined whether DNA–histone complexes (DHC) binding to CPAbs, before cell entry, could modify the cell penetration of CPAbs or their nuclear staining properties. By applying confocal microscopy and image analysis, we found that extracellular binding of purified CPAbs to DHC significantly enhanced their subsequent cell‐entry, both in terms of percentages of positively labelled cells and fluorescence intensity (internalized CPAb amount), whereas there was a variable effect on their nuclear staining profile. Internalization of CPAbs, either alone or bound to DHC, remained unaltered after the addition of endocytosis‐specific inhibitors at 37° or assay performance at 4°, suggesting the involvement of energy‐independent mechanisms in the internalization process. These findings assign to CPAbs a more complex pathogenetic role in systemic lupus erythematosus where both CPAbs and nuclear components are abundant.

Anti‐DNA cell‐penetrating autoantibodies have been extensively studied in autoimmune but not in normal sera. We investigated herein the presence and properties of cell‐penetrating antibodies (CPAbs) in intravenous immunoglobulin (IVIg), a blood product of pooled normal human IgG. IVIg cell penetration was observed into various cell lines, as well as cells from several organs of mice injected intravenously with IVIg therapeutic dose. In all cell types examined in vitro and in vivo, intracellular IgG localized in the cytoplasm, in contrast to the nuclear accumulation of disease‐related CPAbs. IVIg was found to rapidly enter cells via an energy‐independent mode. The CPAb‐fraction was isolated and found to be polyreactive to nuclear and cytoplasmic components; although it corresponded to ~2% of IVIg, it accounted for its inhibitory effect on splenocyte activation. Investigation of IVIg cell penetration capacity provides insight into its mechanisms of action and may account for some of its beneficial effects in numerous diseases.

Natural autoantibodies (autoNAb) recognize self antigens and are an important component of the immune system, as species ranging from invertebrates to vertebrates have polyreactive IgM NAbs. In higher vertebrates, different polyreactive autoNAbs isotypes are also frequently encountered and autopolyreactive IgG NAbs are largely predominant compared to low-titer monoreactive IgG NAbs specific for either self or non-self antigens. Autopolyreactive NAbs manifest the capacity to recognize three-dimensional structures and thus represent a fundamental feature of the immune system that has long been preserved during evolution. NAbs are produced in a continuum of functional and phenotypic tiers of B cells and are likely to derive from proteins initially selected to build the organism that were adapted through evolution to recognize environmental constituents, while preserving their capacity to recognize self antigens. The clonal selection is considered the predominant mechanism of the regulation of the immune system complexity but growing evidence suggests that autoNAbs are also actively implicated. In all species NAbs reacting with either self or non-self antigens constitute a vast network of infinite interactions providing high complexity, stability and plasticity. This evolutionary process was intended to allow the effective recognition of environmental antigens, immune memory, immunoregulatory phenomena, as well as tissue homeostasis. The present article is intended to illustrate the history and the current and future developments in our understanding of self and non-self recognizing NAbs to ultimately enlighten the complexity of the immune system regulation.

Several matters concerning the term "Autoimmunity" have arisen the last two decades. Most researchers agree that a degree of natural autoimmunity in the absence of disease is needed for the development of effective immune responses against infectious agents or cancer cells. Individuals, however, with suitable genetic background and after exposure to certain environmental triggers (such as UV radiation, bacteria, viruses, etc) may develop an exaggerated immune response against self leading to the development of several autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus etc. In this context, a the meeting on "Autoimmunity: Physiological and Pathophysiological Aspects" was held on May in Athens, Greece aiming to bring together and discuss different points of view of the principal investigators that have contributed in the development of this field during the last years. Several aspects of both natural and pathological autoimmunity as well as the possible links between these two states are presented by leading authorities of the field in this special issue.

In over one century of research in immunology marked progress in the scientific knowledge and the implications derived from it has been made. At the same time several contradictory and seemingly opposing results have been obtained. The term autoimmunity is still conceived by many as a term directly related to an immunopathological state. However, strong evidence exist that not only the immune system is able to recognize self-constituents, but it appears also that this property is essential for homeostasis. Direct or indirect alterations of such self-recognition properties of the immune system may contribute to pathology. In this review, the most recent advances in the field of naturally occurring B-cell autoreactivity in health as well as in disease are presented and discussed.

Colleagues have brought to my attention the article “Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)” by R.M. Lequin, recently published in Clinical Chemistry (1 …

Rat spleen lymphocytes were iodinated (125I) with lactoperoxidase. Quantitative autoradiographic studies on cells fixed immediately after iodination showed 1924% of intracytoplasmic grains at 3HD and over from the plasma membrane. Normalization of grain density distribution and comparison of resulting curves with the universal curve of grain scatter of 125I showed that a significant percentage of intracytoplasmic grains (36%) originates from intracytoplasmic labeled sources rather than from scattering from the heavily labeled plasma membrane. Damaged ceils had a threefold grain density than intact cells. Radioactivity counts in sliced 1glyacrylamide gels of iodinated cells revealed 65-72% of total radioactivity in five peaks of apparent tool wt of 44, 50, 57, 90 and 195 thousand daltons. Segregation and internalization of anti-immunoglobulin-Ig-horseradish peroxidase (HRP) complexes from the iodinated plasma membrane proteins of lymphocytes was studied with quantitative autoradiography (1251) and peroxidase cytochemistry; 64% of grains at 1.5HD (1,500 A) from the plasma membrane were within the cap zone, and 36% of grains remained outside the capped immunoglobulins; 45-57% of grains internalized together with Fabanti-Ig-Ig-HRP, and 68% of grains internalized together with anti-Ig-Ig-HRP. These studies indicate that (a) iodination of rat spleen lymphocytes results in a significant internal labeling and that (b) immunoglobulins segregate into caps and internalize together with other iodinated plasma membrane proteins while a significant percentage of iodinated proteins (36%) are excluded from the immunoglobulin caps or internalization sites (32-55%). In 1971, Taylor et al. (31) reported that plasma membrane (surface) immunoglobulins of lymphocytes incubated with fluorescein-labeled anti-immunoglobulin antibodies aggregate into patches, form a polar cap, and undergo endocytosis (internalization). Energy and temperature (22-36~ are necessary for capping and endocytosis of plasma membrane immunoglobulins (7, 20, 25, 33, 34). Capping of anti-immunoglobulin-immunoglobulin complexes is independent of cell moveTHE JOURNAL OF CELL BIOLOGYVOLUME 70, 1976. pages 477-493 477 on A uust 6, 2017 jcb.rress.org D ow nladed fom merit (34). Endocytosis of surface immunoglobulins of lymphocytes and plasma cells has been observed after incubation with monovalent (Fab) antibody fragments, but capping of surface immunoglobulins requires previous incubation with the bivalent antibody (2, 7). Immunoglobulins as well as several other surface antigens of lymphocytes show redistribution and endocytosis after exposure to appropriate antibodies or ligands (4, 6, 13, 24, 25, 26, 37); fur thermore, certain plasma membrane antigens segregate together after exposure to antibody against either one of the antigens, while other antigens move quite independently (6, 24, 25). These experiments have suggested that plasma membrane proteins of lymphocytes segregate from the rest of the membrane and move independently through the fluid matrix of the plasma membrane (27). It has not yet been estabDished whether (a) the segregation and the internalization of plasma membrane Ig is induced by the anti-lg antibody or whether (b) other plasma membrane proteins are associated with the migration of the Ig molecules. In order to gain further information regarding these two questions, we have examined with ultrastructural methods the segregation of plasma membrane immunoglobulins (lg) from lactoperoxidase-iodinated plasma membrane proteins. Lactoperoxidase is a nonpermeant probe which catalyzes the iodination of exposed membrane proteins of intact cells, and has been used for biochemical studies of plasma membranes (11, 16, 17, 21,23~ 29, 32, 35). Since a systematic quantitative ultrastructural autoradiographic study of lactoperoxidase-iodinated cells is not available, we have performed this study, with the methods described by Salpeter, on cells fixed for autoradiography immediately after iodination or after incubation with anti-lg antibody (8, 9, 12, 17, 21, 23, 28). Rat spleen cells iodinated with lactoperoxidase were incubated with conjugates of anti-lg antibody and horseradish peroxi-

We have previously observed that aged lupus-prone (NZB/NZW)Fl (BWF1) mice when infected with Plasmodium chabaudi show an improvement in their clinical lupus-like symptoms. In order to study the mechanisms involved in the long-lasting protective effect of the P. chabaudi infection in lupus-prone mice we analysed specific aspects of the cellular response, namely the profiles of cytokine mRNA expression and cytokine secretion levels in old BWF1 mice, in comparison with uninfected age-matched BWF1 mice and infected or uninfected BALB/c mice. Two months after infection, cells from BWF1 mice were stimulated with concanavalin A (Con A) and demonstrated a recovery of T cell responsiveness that reached the levels obtained with BALB/c cells. Old BWF1 mice showed high levels of interferon-gamma (IFN-gamma) and IL-5 production and correspondingly low levels of IL-2 and IL-4 secretion before infection with P. chabaudi. Infection did not modify the IFN-gamma levels of BWF1 T cells, whereas it considerably increased the secretion of the Th2-related cytokines IL-4, IL-5 and IL-10. In addition, only BWF1 T cells showed increased mRNA expression of tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta). This counter-regulatory cytokine network of infected BWF1 mice may be involved in the improvement of their lupus symptoms. The results of our investigations using the complex model of P. chabaudi infection can be extended and, by using more restricted approaches, it may be possible to explain the multiple regulatory defects of lupus-prone mice.

In lupus‐prone (NZB×NZW)F 1 (B/W) mice, elevated levels of polyreactive autoantibodies bearing the D23 idiotype (Id), characteristic of natural antibodies, were detected before and after the appearance of pathological anti‐DNA antibodies. While these D23 Id + antibodies were able to regulate anti‐DNA antibodies in the early stage of the disease, we found that during disease evolution they had lost their normal ability to regulate anti‐DNA antibodies and furthermore could participate in the lupus‐like syndrome. To explore further the role of the D23 Id + antibodies, we injected young B/W mice with a peptide corresponding to the V H CDR3 region of the D23 monoclonal natural antibody (mNAb). High levels of monospecific antipeptide, as well as polyreactive antibodies, were induced. Among them, the most markedly enhanced antibody population was DNA‐reactive immunoglobulin G1 (IgG1). Compared with controls, these immunized mice had a delayed 50% survival rate and proteinuria developed later. Furthermore, IgG1 able to react with IgG2a anti‐DNA monoclonal antibodies derived from B/W mice were also produced after peptide immunization. Thus, a peptide corresponding to the CDR3 of the D23 mNAb antibody might play a role in the regulation of murine lupus.

Natural, often autoreactive antibodies are present in normal sera in large quantity and show alterations in specificity in diverse pathological situations. They have, however, usually not been studied longitudinally. Here we investigated some representative serum reactivities of natural antibodies in 67 normal children and 10 with injury during childhood, followed up for 3 years. Normal children showed an individually characteristic and relatively stable level of most IgM, IgG, and IgA reactivities when measured with ELISA by reference to a standard. Injured children showed some very rapidly enhanced reactivities within 3 days after trauma, which thereafter slowly diminished over years before coming back to a normal level. This period exceeds by far the lifetime of antibodies and plasma cells. We conclude that natural antibodies contribute to the establishment and maintenance of immune memory in a manner that is distinct from classical immune reactions.

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.