Disclosed herein is a method for following up a prognosis of children with autism before and after treatment with different modalities administered by their clinicians, confirming the involvement of infectious agents, dietary proteins, and toxic chemicals in development of autism. The method utilizes detection of increased amounts of antibodies against an antigen based on infectious agent, toxic chemicals, or dietary proteins. Another method utilizes detection of antibodies to a self-tissue or peptide.

SUMMARY OF THE INVENTION

An embodiment provides for a method for determining etiology of an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of at least one infectious agent derived antigen or antibody against an infectious agent derived antigen, at least one toxic chemical derived antigen or an antibody against a toxic chemical, and at least one dietary protein derived antigen or antibody against a dietary protein, in one or more samples from the patient; b) comparing the level of antigens and/or antibodies determined in step a) with a normal level of the antigens and/or antibodies from control subjects, wherein (i) normal level or lower than normal level of antigens and/or antibodies for the each of said antigens indicate absence of an etiology of autistic spectrum disorder from presence of said antigens; and (ii) higher than normal level of antigens and/or antibodies for one or more of said antigens and/or antibodies indicates a likelihood of the autistic spectrum disorder being based on the presence of said antigens.

Another embodiment provides for a method for determining etiology of an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of antibodies to a self-tissue or peptide in one or more samples from the patient; and b) comparing the level of antibodies determined in step a) with a normal level of the antibodies from control subjects, wherein (i) normal level or lower than normal levels of antibodies indicate absence of etiology of autistic spectrum disorder from presence of said antibodies; and (ii) higher than normal level of the antibodies indicates a likelihood of the autistic spectrum disorder being based on the presence of said antibodies.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Similar to many complex diseases (10), genetic and environmental factors including infections, xenobiotics, dietary proteins and peptides, play a role in the development of autism. The effects of environmental factors on genetic makeup can result in immune, gastrointestinal, neurological, biochemical and neuroimmunological abnormalities. Based on extensive research (11-15), we postulated that autism is induced by infectious agent antigens, toxic chemicals or dietary proteins. This process begins in the gastrointestinal tract but manifests itself in the brain (FIG. 1). These factors will be explained in detail in the following sections.

An embodiment provides for a method for determining etiology of an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of at least one infectious agent derived antigen or antibody against an infectious agent derived antigen, at least one toxic chemical derived antigen or an antibody against a toxic chemical, and at least one dietary protein derived antigen or antibody against a dietary protein, in one or more samples from the patient; b) comparing the level of antigens and/or antibodies determined in step a) with a normal level of the antigens and/or antibodies from control subjects, wherein (i) normal level or lower than normal level of antigens and/or antibodies for the each of said antigens indicate absence of an etiology of autistic spectrum disorder from presence of said antigens; and (iii) higher than normal level of antigens and/or antibodies for one or more of said antigens and/or antibodies indicates a likelihood of the autistic spectrum disorder being based on the presence of said antigens.

Another embodiment provides for a method for determining etiology of an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of antibodies to a self-tissue or peptide in one or more samples from the patient; and b) comparing the level of antibodies determined in step a) with a normal level of the antibodies from control subjects, wherein (i) normal level or lower than normal levels of antibodies indicate absence of etiology of autistic spectrum disorder from presence of said antibodies; and (ii) higher than normal level of the antibodies indicates a likelihood of the autistic spectrum disorder being based on the presence of said antibodies. Preferably, the higher than normal level of antibodies is calculated by taking a mean of levels of antibodies in individuals without symptoms relating to autistic spectrum disorder. Preferably, the higher than normal levels of antibodies is higher than about two standard deviations of normal level of antibodies of a control group.

Preferably, the determining the level of antibodies is accomplished using an immunoassay, such as ELISA, RAST, dot blot, Western blot, and ELISPOT. Preferably, the antibodies used in the immunoassay is selected from IgG, IgA, or IgM.

As used herein, "autistic spectrum disorder" refers to a developmental disorder that affects many aspects of a child's functioning. Autistic spectrum disorder can include, but is not limited to, autism, pervasive developmental disorder, and Asperger's Syndrome. Autistic spectrum disorder can occur in combination with other disorders, such as Attention Deficit Hyperactivity Disorder (ADHD) (which is part of the pervasive developmental disorder), learning disabilities (LD), anxiety disorders, obsessive-compulsive disorders (OCD), epilepsy, or mental retardation.

As used herein, "derived" or "derivative" refers to anything obtained or deduced from another.

The Role of Infectious Agents in Autism

Many infectious agents, including Streptococcus, measles, Rubella, Cytomegalovirus, Varicella zoster, Herpes type-6 and others have long been suspected as etiologic factors in autism (2-4, 16-18). Maternal or post-maternal exposure to these infectious agents may result in neurological disorders including autism. Using the observation that maternal infection increases the risk of schizophrenia anal autism in offspring, recently it has been shown that respiratory infection of pregnant mice (both BALB/c and C57BL/6 strains) with the human influenza virus resulted in offspring that displayed highly abnormal behavioral responses as adults. As in schizophrenia and autism, these offspring displayed deficits in prepulse inhibition (PPI) in the acoustic startle response. Compared with control mice, the infected mice also showed striking responses to the acute administration of antipsychotic and psychomimetic drugs. Moreover, these mice were deficient in exploratory behavior in both open-field and novel-object tests, and they were deficient in social interaction. At least some of these behavioral changes were likely attributable to the maternal immune response itself. They concluded that abnormal levels of cytokine production, which interfere with neuroimmuno-communications, are responsible for abnormal development of the brain (19-20).

Another explanation for disease development postulates that specific antigenic epitopes from an unspecified infectious agent or agents induce(s) a host immune response in which cross-reactivity with myelin triggers disease, a concept referred to as molecular mimicry. In this scenario, certain T-cells and/or antibodies elicited in response to antigens of the infectious agent also recognize relevant self-antigens in the CNS, thereby initiating the destructive autoimmune process (11-15, 21-27).

Infectious Agents and Response to Vaccinations

Many infectious agents, including measles, rubella virus and Cytomegalovirus, Herpes Type-6 and anaerobe bacteria such as Clostridum difficile have been implicated in autism. Therefore, the detection of nucleic acids and antibodies in blood may indicate ongoing infection and justify treatment with anti-bacterial or anti-viral agents (77-80). Moreover, measurements of antibodies against measles, mumps, rubella (MMR), diphtheria, pertusis, tetanus (DPT) and Hepatitis B will assess immune response to immunization and production of protective antibodies. Moderate elevation in IgG antibody against the components of MMR, DPT and Hepatitis B vaccines indicate optimal immune response and good immunological memory to these bacterial and viral antigens. High or very high levels of IgG antibodies against antigenic components of the vaccines indicate overactive immune response against them. Low levels or absence of IgG antibodies against components of vaccines may indicate lack of immunological memory and possibly immune deficiency in the immunized individual.

Examples of antibodies associated with infectious agents and response to vaccinations to be tested include, but are not limited to, measles, mumps, rubella, diphtheria toxoid, pertussis, tetanus toxoid, hepatitis B, herpes type 6, and clostridium neurotoxin.

The Role of Heavy Metals and Other Toxic Chemicals in Autism

Xenobiotics or toxic chemicals have been suspected to contribute to the induction of autoimmunity (30-34). Many environmental chemicals or drugs are toxic to hosts, and their detoxification is achieved primarily in the liver. During their metabolism, they may form reactive metabolites, which can then modify cellular proteins to form neoantigens. The precise mechanisms that lead to modification of self-proteins and the molecular requirements for this modified self to induce tolerance breakdown remain to be established. However, it is important to note that the direct toxic effect of xenobiotics is usually dose dependent and may be evident in the majority of individuals shortly after drug intake; hence, they are relatively easy to identify. In contrast, the immune-mediated effects that follow the intake of drugs or xenobiotics may take a prolonged period of time to be clinically manifest, making the identification of the causative agents a formidable task (35).

Edelson and Cantor (5, 36) demonstrated that neurotoxicants play a possible role in more than 90% of autistic children. These authors presented evidence for genetic and environmental aspects of a proposed process involving immune system injury and autoimmune responses secondary to exposure to immunotoxins. They believe that activation of the immune system is caused by toxicants leading to the production of autoantibodies against haptens, i.e., the toxic chemicals attached to brain proteins. The subsequent damage may be considered a component in the etiologic process of neurotoxicity in the autistic spectrum.

For a chemical compound to lead to an autoimmune response, it is generally thought that the compound must first become covalently bound to a carrier protein (37, 39). Immune reactions to drugs or their metabolites can develop when a hapten carrier complex interacts with gut-associated lymphoid tissues (GALT) that constitute the largest lymphoid organ (38). If covalent adducts of drugs or other chemical compounds are formed in GALT, it seems reasonable that they may lead to immune responses and chemically-induced Type I- Type IV allergic reactions (37). In fact, the non-steroidal anti-inflammatory Dicoflenac has been shown to cause a variety of idiosyncratic adverse reactions such as hemolytic anemia, hepatotoxicity, agranulocytosis, and anaphylaxis, all of which are components of immune reactions to protein adducts of Diclofenac (37-39). These protein adducts can be formed by direct reaction with tissue antigens or cytochrome P450 dependent and UDP-glucuronosyltransferase dependent pathways of metabolism. For example, immunoblot analysis of small intestine homogenates and isolated enterocytes with drug-specific antiserum revealed protein adducts of diclofenac. Two of these adducts of Diclofenac were identified as aminopeptidase N (CD 13) and sucrase-isomaltase (38). Intestinal protein adducts of chemicals can, therefore, be formed in GALT where they may lead to allergic reactions, inflammation and autoimmunity.

Among many toxicants, such as thimerosal, merthiolate, ethyl mercury, or other mercury-based compounds, in vaccines has been associated with immune injuries described in children with autism (41-44). Contrary to many haptens that bind covalently to a single amino acid, such as lysine, metal complexes consist of a central metal ion composed of four different amino acids, and hence they possess increased complex stability (37). To demonstrate possible binding of ethyl mercury to DPP IV and CD69, we postulated that in addition to infectious agent antigens such as Streptokinase, ethyl mercury (xenobiotic) binds to different lymphocyte receptors and tissue antigens. We assessed this hypothesis first by measuring IgG, IgM and IgA antibodies against CD26, CD69 and SK against ethyl mercury bound to human serum albumin in patients with autism. A significant percentage of children with autism developed anti-SK, and anti-ethyl mercury antibodies, concomitant with the appearance of anti-CD26 and anti-CD69 autoantibodies. These antibodies are synthesized as a result of SK and ethyl mercury binding to CD26 and CD69, indicating that they are specific. Immune absorption demonstrated that only specific antigens, like CD26, were capable of significantly reducing serum anti-CD26 levels. However, for direct demonstration of SK and ethyl mercury binding to CD26 or CD69, microtiter wells were coated with CD26 or CD69 alone or in combination with SK or ethyl mercury and then reacted with enzyme labeled rabbit anti-CD26 or anti-CD69. Adding these molecules to cD26 or CD69 resulted in 28-86% inhibition of CD26 or CD69 binding to anti-CD26 or anti-CD69 antibodies. We, therefore, propose that bacterial antigens and thimerosal (ethyl mercury) in individuals with pre-disposing HLA molecules, bind to CD26 or CD69 and induce antibodies against these molecules as well as to lymphocyte receptors and tissue antigens, resulting in autoimmune reaction in children with autism.

Neuroimmune Abnormalities Induced by Xenobiotics and Metals

It is of considerable interest that antibodies to neuron-specific antigens are prevalent in populations exposed to environmental and occupational chemicals and in patients with neurodegenerative diseases in which viruses or other infectious agents are the suspected etiological agents. For example, IgG antibodies to MBP, neuronal cytoskeletal proteins and neurofilaments are detected in workers exposed to lead or mercury (45). The titer of these antibodies is significantly correlated with blood lead or urinary mercury, which are the typical indices of exposure. Moreover, the level of these antibodies is correlated with the degree of sensorimotor deficits, because these antibodies interfere with neuromuscular function (46).

Taking into consideration the regulatory interactions between the nervous system and the immune system, as well as the detection of MBP and NAFP autoantibodies, it is therefore quite plausible to propose that drugs and environmental toxins might have detrimental effects on neuroendocrine-immune circuits, thereby resulting in autism. Toxic chemical exposure to substances, such as polychlorinated biphenyl, mercury, lead and other similar potentially harmful agents may induce alteration or over-expression of the genes involved in regional brain glial fibrillary acidic protein (GFAP) and astroglial glucose regulated protein (GRP). The astroglial cytoskeletal element GFAP, neurotypic and gliotypic proteins or neurofilament triplet are generally accepted as sensitive indicators of neurotoxic effects in mature brains (47, 48).

Over-expression of the gene results in altering the structural differentiation of astrocytes and the subsequent autoimmune response to neurofilaments and astroglial glucose regulated proteins. Autoantibodies against neurological antigens in autism have been studied in our laboratory extensively and found to be elevated in children with autism (11-15). The high prevalence of these autoantibodies in neurodegenerative and neuropsychiatric disorders has led many investigators to believe that these antibodies reflect an alteration of the blood-brain barrier, which promotes the access of immunocompetent cells to the central nervous system (49-52).

In these studies, we were able to present viable evidence in support of the genetic and environmental aspects of a hypothetical process believed to cause immune system injury secondary to immunotoxins exposure. Activation of the immune system is caused by toxicants, leading to the production of autoantibodies against haptens--the toxic chemicals attached to brain proteins. The resulting damage may be considered a component in the etiologic process of neurotoxicity in the autistic spectrum.

Autoimmune Reaction Induced by Heavy Metals

Mercury is a widely distributed environmental and industrial pollutant. This is why methyl mercury is often detected in many fish. In fact, ethyl-mercury or thimerosal has been used in increasing amounts as preservatives in many vaccines since the 1950's. Therefore, during the first year of life when the immune system is in the process of maturation, children become exposed to up to 100 micrograms of mercury, which greatly exceeds the CDC threshold. Exposure to large doses of mercury results in acute renal tubular lesions and immunosuppression, whereas chronic administration of smaller doses can lead to development of systemic autoimmunity (31-34). The characteristic features of mercury-induced autoimmunity are very similar to manifestations of SLE. This includes: increased levels of Class II MHC antinuclear antibody production hypergammaglobulinemia polyclonal antibody to self-antigens formation of immune complexes lymphocyte proliferation necrotizing vasculitis

Mercury-induced autoimmunity is also similar to lupus in that the disease process requires CD4+T-cells, T- and B-cell stimulatory molecules and interferon-.gamma., which strongly suggests identical pathogenic mechanisms. Given the complexity of metal, interaction with cellular and subcellular components of the immune system and the large number of molecules that may be affected, genetic studies were initiated to define the genes responsible for sensitivity of resistance to mercury-induced autoimmunity. A single major quantitative trait locus on chromosome 1, designated as Hm.gamma.1 was linked to glomerular immune complex deposits (81). Mercury is only one of a number of immunostimulatory heavy metal xenobiotics that can induce adverse immunotoxicity. Several of these such as silver or gold also promote the production of anti-fibrillarin autoantibodies only in mercury-sensitive mouse stains (82).

Indeed after injection of methyl-mercury, a number of murine strains develop an antibody response against U3 small nucleolar ribonucleo-proteins called fibrillarin and chromatin. These antibodies have also been detected in humans with scleroderma. Therefore, detection of anti-nuclear antibody along with metals, fibrillarin and chromatin antibodies and elevation in immune complexes indicate involvement of metals in induction of inflammation and autoimmunity in autism (82). Further, production of these antibodies may indicate a lack of functional metallothionein at cellular level.

Examples of antibodies associated with autoimmune reaction and involvement of metals to be tested include, but are not limited to, anti-nuclear protein, mercury, fibrillarin, chromatin, immune complexes, and metallothionein.

Neuroimmune Antibodies Induced by Dietary Proteins and Infectious Agents

As mentioned above, many infectious agents have long been suspected of being etiologic factors in autism. Whether or not these viruses actually induce brain autoantibodies has not yet been explored. For this reason, we decided to review the available scientific literature and found that over sixty different microbial peptides have been reported to cross-react with human brain tissue and MBP. Furthermore, these peptides not only have the capacity to cross-react with MBP and induce T-cell response, but also are also able to induce experimental autoimmune encephalomyelitis (11, 26-30).

Among families with autistic children, it is well known that the elimination of milk from the child's diet significantly improves the patient's condition. Investigators found that an encephalitogenic T-cell response to MOG can either be induced or alternatively suppressed as a consequence of immunological cross-reactivity or molecular mimicry with the extracellular IV-like domain of milk protein butyrophilin. All of these clinical laboratory findings shed light on our detection of higher levels of antibodies against milk antigens in autistic sera. Based on earlier publications, we chose Streptococcus synthetic peptide containing the conserved M protein or brain crossreactive epitope, a Chlamydia pneumoniae-specific peptide and the butyrophilin milk peptide, which modulates the encephalitogenic T-cell response to MOG in experimental autoimmune encephalomyelitis for our cross-reactivity study (11, 65).

Detection of IgG, IgM and IgA antibodies against myelin basic protein, neurofilaments and their cross-reactive epitopes in milk, Streptococcus and Chlamydia may justify treatment with antibiotic and/or elimination diet.

Examples of neuro-autoimmune antibodies induced by dietary proteins and infectious agents and antibodies associated with the neuro-autoimmune antibodies to be tested include, but are not limited to, myelin basic protein, neurofilament, milk butyrophilin, streptococcus M protein, and chlamydia pneumoniae.

Binding of Dietary Peptides to Different Tissue Enzymes May Promote Development of Peptidase Antibodies in Children with Autism

Opioid peptides are available from a variety of food sources. These dietary proteins and peptides, including casein, casomorphins, gluten (GLU) and gluteomorphins, can stimulate T-cells, induce peptide-specific T-cell responses, and abnormal levels of cytokine production, which may result in inflammation, autoimmune reactions and disruption of neuroimmune communications (54-57). In celiac disease (CD), a majority of patients who express HLD-DQ2 and/or DQ8 react to a 33-mer peptide and 15 other T-cell stimulatory peptides (58, 59). This peptide binding to HLA-DQ2 and HLA-DQ8 molecules is most efficient when negatively charged amino acids are present at anchor positions in the peptide. Yet GLU contains very few negatively charged amino acids, which makes GLU-derived peptides low affinity ligands for HLA-DQ2 and -DQ8. This paradox has been solved by finding that enzyme tissue transglutaminase, target of endomysium-specific antibodies in CD patients, can modify GLU peptides by conversion of glutamine residues into glutamic acid, which introduces negative charges favored for binding (58-60).

A majority of children with autism cannot tolerate wheat and milk proteins or peptides and hence elimination of these peptides from the diets significantly improves their conditions. This clinical finding correlates with laboratory results reported earlier by our group in children with autism (11-15) and by different investigators in MS-like syndromes (61-64). They found that an encephalitogenic T-cell response to myelin oligodendrocyte glycoprotein (MOG) could be either induced or alternatively suppressed as a consequence of immunological cross-reactivity or "molecular mimicry" with the extra-cellular IV-like domain of milk protein called butyrophilin (BTN) (65). We detected IgG, IgM and IgA antibodies against nine specific neuron-specific antigens in the sera of children with autism. These antibodies were found to bind with different encephalitogenic molecules that have sequence homologies to a milk protein (11).

Indeed, when we tested IgG, IgM and IgA antibodies against milk peptides, we found that every single serum with ELISA values higher than 0.3 O.D. against neurological antigens also exhibited high levels of antibodies against neurological antigens and antibodies against milk peptides in a higher percentage of experimental sera. Similar to milk peptides, antibodies against different gliadin peptides have also been described in celiac disease and gluten ataxia (66-67).

Food Allergies and Intolerance

Food allergies may be said to be contributory to the behavioral disorders of individuals inflicted in autism. Sensitivity to gluten and milk are thought to be the major food allergens in these patients. In one study, nineteen children with autistic syndromes were treated with either gluten-free milk and milk-reduced diets, or milk-free and gluten-reduced diets. Before treatment, five of the fifteen fully studied patients had increased levels of IgA antibodies to casein or gluten. After following the diet for a year, improvement was noted in terms of increased social contact, decreased stereotypy, an end to self-mutilation (like head banging), and a decrease in "dreamy state" periods. These improvements were accompanied by a significant decrease in urinary peptide excretion. The possible mechanism is that children with autism suffer from one or more peptidase defects that fail to break down "exomorphins" (exogenous opioids) found in milk and wheat. These exorphin peptides then gain entry into the brain where they significantly disrupt brain chemistry (see FIGS. 2, 3).

The presence of other food allergies should also be determined, as food allergies are likely the factor responsible for the increased intestinal permeability noted in these patients. In fact, increased gut permeability has been suggested as a possible causative factor for autism (73, 77).

Examples of antibodies associated with food allergy and intolerance to be tested include, but are not limited to, milk, casomorphin, wheat gluten/gliadin, gluteomorphin, corn, soy, and tissue enzyme, such as transglutaminase which may modify resultant dietary peptides.

Cross-Reaction Between Gliadin and Cerebellar Purkinje Cells as a Possible Mechanism for Neuroimmune Abnormalities in Autism

One of the most frequent presentations of gluten sensitivity is the neurologic dysfunction called gluten ataxia. Up to 33% of patients presenting with neurologic dysfunction and 90% of patients presenting with pruritic vesicular rash of dermatitis herpetiformis associated with gluten sensitivity also have celiac disease (66). While the remaining patients have serologic markers or anti-gliadin antibodies and genetic susceptibility (HLADQ2), they do not have histologic evidence of small bowel involvement. Based on a major epidemiologic study involving more than 200 patients, gluten ataxia was found to account for 40% of cases with idiopathic sporadic cerebellar degeneration. When patients with gluten ataxia were autopsied, perivascular cuffing with inflammatory cells, predominantly affecting the cerebellum, and loss of Purkinje cells were detected. These inflammatory reactions resulting in Purkinje cell loss imply that the neurologic insult may be immune-mediated (67, 69, 70). It is not clear whether such immune-mediated damage is primarily cellular or antibody-driven. In a recent study, investigators assessed the reactivity of sera from patients with gluten ataxia, patients newly diagnosed with celiac disease without neurologic dysfunction and healthy control subjects (67).

Using indirect immunocytochemisty on human cerebellar and rat CNS tissue, cross-reactivity of a commercial IgA antigliadin antibody with cerebellar tissue was analyzed. Sera from 12 of 13 patients with gluten ataxia strongly presented stained Purkinje cells. Less intense staining was observed in some but not all sera from patients with newly diagnosed celiac disease without neurologic dysfunction. At high dilutions (1:800) staining was observed only using sera from patients with gluten, ataxia but not from control subjects. Sera from patients with gluten ataxia also stained some brainstem and cortical neurons in rat CNS tissue. Commercial anti-gliadin antibody stained human Purkinje cells' in a similar manner. Absorption of the antigliadin antibodies using crude gliadin abolished the staining in patients with celiac disease without neurologic dysfunction, but not in those with gluten ataxia. The conclusion suggested that patients with gluten ataxia have antibodies against Purkinje cells that cross-react with epitopes on Purkinje cells, and humoral immune responses are involved in the pathogenesis of gluten ataxia (67).

Direct Evidence for Structural Similarity Between Gliadin Peptides and Cerebellar Antigens

Several distinctive neurologic disorders occur in patients with paraneoplastic cerebellar degeneration (PCD). The syndrome of PCD is among the most common of these disorders and generally occurs in patients with neoplasms of the lung, breast, ovary, or with Hodgkin's disease. Neuropathologic features of PCD include extensive loss of Purkinje cells, degenerative changes in the remaining Purkinje cells, as well as variable losses of granule and basket neurons.

The presence of anti-Purkinje cell antibodies in some PCD patients suggests an autoimmune etiology. To identify the molecular targets for these autoantibodies, an Agt11 cDNA expression library from human cerebellum was constructed and screened with IgG from a patient with paraneoplastic cerebellar degeneration. A single clone, pCDR2, produced a fusion protein that reacted strongly with the patient's IgG. Sequencing the pCDR clones revealed 6 amino acids repeated in tandem along the entire cDNA sequence (VAL, PRO, LEU, LEU, GLU, ASP). (SEQ ID NO: 4). This gene was expressed predominantly in neuroectodermal tissues (68).

Neurotransmitters and Neuroimmune Miscommunication

Autism was originally thought to be primarily a psychiatric condition. However, recent biochemical genetic studies have lead to the hypothesis that the disorder is due to an organic defect in brain development. Specifically, autism is thought to be a result of abnormal serotonin metabolism in the brain. The abnormalities that have been documented include:

abnormal release and uptake of serotonin by platelets

abnormal kynurenine metabolism

increased serum serotonin and free tryptophan levels

abnormal urinary 5-hydroxyindolacetic acid (5-HIAA) levels

abnormal urinary Serotonin metabolites

The basic defect appears to be a decrease in CNS Serotonin activity despite elevated free tryptophan levels in the serum. Abnormal serotonin metabolites seen in autistic children may significantly contribute to their mental dysfunction (83-85). Drugs such as LSD, psilocybin, ergot, and other hallucinogens are serotonin analogs, and a number of serotonin metabolites are known to be hallucinogens. It is also interesting to note that serotonin and its metabolites are produced in, and absorbed from, the intestines.

This abnormal level of serotonin along with reaction of bacterial toxins, xenobiotics and dietary peptides with different aminopeptidases and gut-neuroimmune communications results in autoantibody production against these important tissue enzymes such as somatostatin, vasoactive intestinal peptides. Formation of antibodies against peptidases results in dysfunctional enzymes and accumulation of peptides in the GI tract and in circulation. These dietary peptides in the blood may bind to G protein receptors, cause immune dysfunction and transmigrate across blood, the blood-brain barrier, and activate the local antigen-presenting cells, such as microglia and astrocytes. By reacting to .mu., .gamma., .kappa. opioid receptors on both lymphocytes and nerve cells, dietary peptides such as pro-dynorphins, dynorphins, casomorphins and gluteomorphins may change the level of cytokine and interfere with neuroimmune communication. Therefore, detection of high or low levels of serotonin along with antibodies to serotonin, somatostatin, vasoactive intestinal peptides, DPP IV, pro-dynorphin and dynorphin may indicate disturbance in gut-neuroimmune communication.

Examples of antibodies associated with neurotransmitters and neuroimmune miscommunication to be tested include, but are not limited to, serotonin receptor antibodies, serotonin antibodies, somatostatin antibodies, vasoactive intestinal peptide, prodynorphin, dynorphin, and dipeptidylpeptidase IV.

Pathogenesis and Mechanism of Autoimmunity and Autism

For cross-reactive circulating antibodies to become pathogenic, they must cross the blood-brain barrier. It is now known that permeability of the blood-brain barrier increases after major histocompatibility complex class I expression (Fabry et al, 1994), activated lymphocyte interaction, and change in neuronal cell adhesion molecules (71, 72). Based on review of literature and results reported here, we propose the following chain of events, as shown in FIG. 4, that may explain possible mechanisms of injury in autism: 1. In the course of a lifetime, the body is exposed to infectious agents, which mimic neuron-specific antigens, such as EBV, CMV, HHV-6, HTLV-1, HTLV-2, streptococcus, Chlamydia pneumoniae or even milk and gluten peptides. 2. Pre-existing auto-reactive T-cells are generated by molecular mimicry as a result of contact with dietary proteins and viral, bacterial, and parasitic antigens, which have sequence homologies or matched motifs with auto antigens. 3. Bacterial enterotoxins, viral antigens, and metals, such as mercury and lead, may increase adhesion molecules on brain endothelial cells. Toxic chemicals may also increase leukocyte function-associated antigens on activated T-cells. 4. Pre-existing autoreactive T-cells may transmigrate across the blood-brain barrier and induce the activation of local antigen-presenting cells, such as microglia and astrocytes. 5. By reacting to .mu., .gamma., and .kappa. opioid receptors on both lymphocytes and nerve cells, dietary peptides such as casomorphins, gluteomorphins and others may change the level of cytokine production and interfere with neuroimmune communication (19, 20, 53; FIGS. 2, 3). 6. This neuroimmune miscommunication may result in production of IL-2, INF-.gamma. and TNF-.alpha. by T-helper-I autoreactive cells and TNF-.alpha. by the antigen presenting cells (astrocytes and microglia may result in oligodendrocyte damage and demyelination). 7. As a result of this sequence of events, MBP, MAG, MOG, .alpha., .beta.-crystallin and other antigens are released from neurofilaments and enter the circulatory system. This results in immune reactions, such as the formation of plasma cells with the capacity of producing IgG, IgM and IgA antibodies against neuron-specific antigens. 8. These antibodies may cross the blood-brain barrier and combine with brain tissue antigens to form immune complexes, thus causing further damage to the neurological tissue. The antibodies, along with toxic biological weaponry, such as arachidonic acid and free radicals, can "chew off" neuron myelin and impair electrical transmission between a muscle and the central nervous system. 9. This hypothesis may explain significant differences in levels of pathogenic autoantibodies between control subjects and patients exposed to toxic chemicals and metals (11, 15, 19, 20, 53).

An embodiment provides for a method for determining etiology of an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of at least one infectious agent derived antigen or antibody against an infectious agent derived antigen, at least one toxic chemical derived antigen or an antibody against a toxic chemical, and at least one dietary protein derived antigen or antibody against a dietary protein, in one or more samples from the patient; b) comparing the level of antigens and/or antibodies determined in step a) with a normal level of the antigens and/or antibodies from control subjects, wherein (i) normal level or lower than normal level of antigens and/or antibodies for the each of said antigens indicate absence of an etiology of autistic spectrum disorder from presence of said antigens; and (iv) higher than normal level of antigens and/or antibodies for one or more of said antigens and/or antibodies indicates a likelihood of the autistic spectrum disorder being based on the presence of said antigens.

Another embodiment provides for a method for determining etiology of an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of antibodies to a self-tissue or peptide in one or more samples from the patient; and b) comparing the level of antibodies determined in step a) with a normal level of the antibodies from control subjects, wherein (i) normal level or lower than normal levels of antibodies indicate absence of etiology of autistic spectrum disorder from presence of said antibodies; and (ii) higher than normal level of the antibodies indicates a likelihood of the autistic spectrum disorder being based on the presence of said antibodies.

Preferred self-tissue or peptide include, but is not limited to, tissue and cell antigens, receptors, mediators, enzymes, and neurotransmitters. More specifically, preferred self-tissue or peptide include, but is not limited to, digestive enzymes (pepsin, trypsin, chymotrypsin), aminopeptidase, dipeptidyl peptidase, CD26, DPPI IV, CD13, CD69, transglutaminase, epithelial cells, brush border antigens and enzymes, colon tissue antigens, gastrin, gastrin inhibitory polypeptide, secretin, motilin, enkephelin, substance P, somatostatin, and serotonin. When a preferred self-tissue antigen or peptide is a neurotransmitter or a neurotransmitter receptor, the preferred self-tissue antigen or peptide is selected from the group consisting of serotonin receptor, serotonin, somatostatin, vasoactive intestinal peptide, pro-dynorphin, dynorphin, dipeptidylpeptidase IV, and complex dipeptidylpeptidase IV.
 

Claim 1 of 7 Claims

1. A method for diagnosing an autistic spectrum disorder in a patient, comprising the steps of: a) determining a level of antibodies to a protein selected from the group consisting of an aminopeptidase N (CD13), dipeptidyl peptidase IV (CD26), dipeptidyl peptidase I and CD69, in one or more samples from the patient; and b) comparing the level of antibodies determined in step a) with a normal level of the antibodies from control subjects, wherein (i) normal level or lower than normal levels of antibodies indicate absence of autistic spectrum disorder in said patient; and (ii) higher than normal level of the antibodies indicates the presence of the autistic spectrum disorder in said patient.
 

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