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Clinical findings in autism and relevance of dysfunctional calcium signalling in

     Brain Development
     Motor/Sensory Disturbances
     Blood Brain Barrier
     Immunity and Inflammation
     Gastrointestinal Issues
     Membrane Metabolism
     Oxidative Stress
     Mitochondrial Dysfunction
     Gender Differences

Dysregulating Factors:
     Genetic Factors
     Infectious Agents





Summary of abnormal biomedical findings in autism

Immunity and Inflammation

Abnormal immune findings in autism

Results of numerous studies point to an abnormality of the immune function in autism, as well as active, ongoing inflammation in the GI tract, the brain and the cerebrospinal fluid (CSF).

A recent study by Vargas et al [15546155] investigated the presence of immune activation in postmortem brain specimens and CSF from subjects with autism. The authors found active neuroinflammation in multiple areas of the brain, for example in the cerebral cortex and white matter, and in the cerebellum. A marked migroglial and astroglial activation was also found, as well as the presence of an altered cytokine pattern, with macrophage chemoattractant protein (MCP)-1 and tumor growth factor-beta1 (TGF-beta1) being the most prevalent cytokines. There was also an accumulation of macrophages and monocytes, and a marked absence of lymphocytes and antibodies, pointing toward an innate neuroimmune activation with the absence of adaptive immune system/T cell activation in the brain. In addition, an enhanced proinflammatory cytokine profile was observed in the CSF, including once more a marked increase in MCP-1. These observations resemble findings in other neurological disorders in which elevations in cytokine levels is associated with the pathogenesis of neuroinflammation, neurotoxicity and neuronal injury and subsequent behavioural and cognitive impairments, for example HIV-associated dementia and multiple sclerosis [15288500, 11282546, 16875710, 9852582]. Another study examinig brain tissue of found that proinflammatory cytokines TNF-alpha, IL-6 and GM-CSF, IFN-gamma and IL-8 were significantly increased in the brains of ASD patients compared with the controls. However the Th2 cytokines (IL-4, IL-5 and IL-10) showed no significant difference. The Th1/Th2 ratio was also significantly increased in ASD patients, suggesting localized brain inflammation and autoimmune pathology [19157572]. Further findings suggestive of autoimmune pathogenesis in a subgroup of patients revealed higher levels of antibodies directed against human cerebellar protein extracts [18706993], and high anti-nuclear antibody seropositivity in ASD patients compared to controls. In addition, Anti-nuclear antibody seropositivity had significant positive associations with disease severity, mental retardation and electroencephalogram abnormalities [19135624]. Autistic children were found to have significantly higher serum anti-myelin-associated glycoprotein antibodies than healthy children [19073846].

Another investigation into inflammatory markers in the brain tissue of patients with autisms revealed significantly increased levels of several proinflammatory cytokines (TNF-alpha, IL-6 and GM-CSF, IFN-gamma, IL-8). The Th1/Th2 ratio was also significantly increased in ASD patients, suggesting that localized brain inflammation and autoimmune disorder may be involved in the pathogenesis of ASD [19157572].

Of some interest in this context may be the role of IL-6, a cytokine that is known to modulate cellebral function and enhance neurotoxicity through influencing Purkinje neuron physiology and electrical activity. Animal experiments illustrate that, during early pre and postnatal development, inflammatory cytokine challenge can induce various psychological, behavioral and cognitive impairments [17804539, 16147952, 9852582]. At the same time the expression of many cytokines, including MCP-1, in neurons and glial cells seems to be upregulated by increased intracellular calcium triggered by membrane depolarisation [11102468, 10943723] (see next section).

Various serological findings further confirm the presence of immune system dysregulation and active inflammation in autism - raised levels of proinflammatory cytokines have often been observed in blood of patients with autism, with significant increases of IFN-gamma, IL-6 and TNF-alpha. These results are followed by findings of decreased peripheral lymphocyte numbers, incomplete or partial T cell activation following stimulation, decreased NK cells activity, dysregulated apoptosis mechanisms, imbalances of serum immunoglobulin levels, increased numbers of monocytes and abnormal T helper cell (Th1/Th2) ratio, with a Th2 predominance, and without the compensatory increase in the regulatory cytokine IL-10 [18762240, 16698940, 16360218]. It is of interest to note that, following increased levels of TGF-beta1 in brain specimen as observed by Vargas et al, this cytokine was found to be significantly lower in the blood of adult patients with autism compared to controls [17030376, 18762342].

Another relevant observation is the elevation of cerebrospinal fluid levels of TNF-alpha compared to its serum levels in subjects with autism. The observed ratio of 53.7:1 is significantly higher than the elevations reported for other pathological states for which cerebrospinal fluid and serum tumor necrosis factor-alpha levels have been simultaneously measured [17560496].

It has been suggested that prenatal viral infection might dysregulate fetal immune system, resulting in viral tolerance in autism [139400] (see Infectious_Agents). Studies showing altered T-cell subsets raised suspicions about possibile autoimmune aspects of autism and several studies pointed to association of the risk of autism to immune genes located in the human leukocyte antigen (HLA) [16720216, 15694999]. The immune system uses the HLAs to differentiate self cells and non-self cells and some of them are linked with higher risk of autoimmune disorders. Animal studies show that behavioural changes follow onset of autoimmune disease and can be reversed through immune-suppressive treatments [8559794], and although various autoantibodies to brain antigens have been observed in autism, the results of those studies are often conflicting. In addition to the inconsistent findings the question has been raised as to whether those autoantibodies would be pathogenic contributors or mere consequences of the the disorder. Of note in this context is the finding pointing to possible association of virus serology and brain autoantibodies in autism [9756729]. One study found that children with autism had a significantly higher percent seropositivity of anti-nuclear antibodies. Anti-nuclear antibody seropositivity was significantly higher in autistic children with a family history of autoimmunity than those without such history, and also had significant positive associations with disease severity, mental retardation and electroencephalogram abnormalities (19135624). Autistic children were found to have significantly higher serum anti-myelin-associated glycoprotein antibodies than healthy children. Family history of autoimmunity in autistic children was significantly higher than controls. Anti-myelin-associated glycoprotein serum levels were significantly higher in autistic children with than those without such history (19073846).

A study looking at several markers of concomitant autoimmunity and immune tolerance found highly elevated circulating IgA and IgG autoantibodies to casein and gluten dietary proteins in autism sample compared to controls. Circulating anti-measles, anti-mumps and anti-rubella IgG were positive in only 50%, 73.3% and 53.3% of autistic children previously immunised by MMR, as compared to 100% positivity in the control group. Anti-cytomegalovirus CMV IgG was also investigated and was positive in 43.3% of the autistic children as compared to 7% in the control group (17974154).

One very interesting finding in recent times was the association of genetic polymorphisms related to macrophage migration inhibitory factor (MIF) in individuals with autism [18676531]. MIF is central in host immune reactions/viral clearance and inflammatory responses. MIF favours viral neuroinvasion by compromising the integrity of the blood-brain barrier (see Infectious Agents re viral aetiology of autism). It is very closely linked to MCP-1 (elevated manifold in autism) and other proinflammatory chemokines, and its levels are inversely related to regulatory cytokine IL-10 (low in autism). MIF also plays a central part in gastrointestinal inflammation (see Gastrointestinal), as well as cellular oxidative stress pathways - cysteine mediated redox mechanisms (impaired in autism, see Oxidative Stress). It is also appears to be directly involved in neuronal function via at least one pathway, that of Angiotensin II. Levels of MIF are often suppressed in fever. The expression levels of MIF gene are regulated by calcium-dependent CREB.

In addition to several mutations in genes belonging to major histocompatibility complex (MHC II) region, related to immune function and associated with autism, the involvement of CREB-mediated events in regulation and expression of these genes should be of utmost interest [16730065, 10458754] (see Brain_Development).

Some aspects of calcium signalling in immunity and inflammation

Proinflammatory cytokines/chemokines are protein signals released from a variety of cells in response to bacterial or viral infections or agents that cause physical damage, for example oxalates, in order to attract leukocytes to the site of inflammation. CC chemokines induce the migration of monocytes and other cell types such as NK cells and dendritic cells. One such example is MCP-1, which induces monocytes to leave the bloodstream and enter the surrounding tissue, becoming tissue macrophages.

Many inflammatory processes are mediated through activities of LTCC and elevation in intracellular calcium level is the central element in the activation of brain macrophages. Several calcium channel blockers at therapeutic concentrations were shown to modulate inflammatory processes. Benidipine for example suppressed induction of MCP-1 and IL-8 [15364009]. In another study nifedipine inhibited TNF-alpha-induced reactive oxygen species generation and subsequent MCP-1 [15921025, 15018305]. The activity of LTCC mediates the cyclic stretch-induced inflammatory gene expression in airway smooth muscle and can be inhibited by nifedipine, with the most responsive genes being the ones encoding for COX-2, IL-6, and IL-8 [16339998, 8576944]. The induction of lymphocyte migration by IL-8, IL-1 alpha and IL-1 beta also appears to be dependent on activities of VGCC and can be inhibited by several calcium channel blockers [2686646]. TNF-alpha concentrations are shown to be suppressed in animals pretreated with verapamil or diltiazem, and by nimodipine in cultured monocytes [15661163]. On the other hand calcium entry blockers increase the regulatory interleukin-10 production, shown to be consistently low in autism [9110418].

Of special interest should be several recent observation of reduced responsiveness to stimulation of both B and T lympocytes following elevations of cytosolic free calcium [12805281] (see Infectios_Agents). Although LTCC are mainly expressed in excitable cells, recent evidence points to the involvement of voltage-insensitive dihydropyridine channels in calcium pathways of both B and T lymphocytes [8631746, 9550376, 14981074]. Calcium influx through the plasma membrane of lymphocytes is essential for their activation, proliferation, cytokine secretion and apoptosis [16766050]. Calcium and CREB-mediated upregulation of chemokine receptors mRNA has been observed in T lympocytes, whereby activation of CXCR4 by viral proteins leads to increased viral infectivity [11874984, 14563373].

Furthermore, differences in calcium mobilization are associated with differentiation of naive CD4+ T cells into Th1 and Th2 subsets. Because LTCC are induced during Th2 but not Th1 cell differentiation, it has been suggested that LTCC blockers may be useful in the treatments of Th2-mediated pathologies. One such agent, nicardipine, was able to inhibit the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals, but had no effect on Th1-mediated experimental encephalomyelitis [15100258, 15777162, 14708347, 12721099].

Apoptosis plays an important role in the control of the immune system, and its impairment may be associated with autoimmune responses. Calcium entry through LTCC is thought to play a central role in apoptosis-related processes, including apoptotic body engulfment by dendritic cells.

Alterations in intracellular calcium levels have been implicated in the pathophysiology of immune dysfunction. Elevation of calcium levels play a central role in the regulation of executive functions in activated microglia, the immunecompetent cells in the brain, which have many similarities with macrophages of peripheral tissues and also express a variety of ion channels. Activation of microglia has been observed during infection, inflammation, physical injury, trauma, ischemia, and in a number of neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, and prion disease [12805281, 9914452]. It has been hypothesised that calcium influx via VGCC plays a significant role in the development of neurological disability and white matter damage in the animal experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS), and MS itself. Administration of calcium channel blockers significantly ameliorated EAE in mice, with spinal cord samples showing reduced inflammation and axonal pathology [15296830]. It should also be mentioned in this context that phosphodiesterase (PDEIs) inhibitors have shown inhibiting effect on cytokine production in lipopolysaccharide-stimulated mouse microglia. PDEIs supressed the production of TNF-alpha, as well as IL-I and IL-6. In contrast, the production of IL-10, was upregulated by certain PDEIs, and it was concluded that these agenst may be useful in treating inflammation in the CNS [10335522]. One of the possible explanations for the anti-inflammatory effect of these agents is their regulatory effect on LTCC [7517800].

Mast cells are important for clearance of pathogens and in wound healing, and play a central role in many forms of allergies, like asthma and eczema. Upon stimulation mast cells degranulate, releasing histamine, prostaglandin and many cytokines. Because of their ability to release a wide range of cytokines and other inflammatory mediators, they are thought to have a central role in innate immunity and are implicated in the pathology associated with inflammatory and autoimmune disorders such as rheumatoid arthritis and multiple sclerosis. The activation and release of both plasma cells and mast cell contents is regulated by the influx of calcium, most probably through Cav1.4 LTCC, which are highly expressed in those cells [9357819].

Relative to autism it may be of interest to mention that a specific mechanism has recently been suggested, whereas the crosstalk between chemokine receptors and neuropeptide membrane receptors serves as a bridge between the immune and nervous systems. Chemokine receptors, a family of G protein-coupled receptors, are widely expressed by cells of immune and nervous systems, including neurons. The activation of several other receptors, such as opioid, vasoactive intestinal peptide, or adenosine receptors, often has inhibitory effects on chemokine receptors by a mechanism termed heterologous desensitization, resulting in suppression of immune responses. Conversely, activation of chemokine receptors also induces desensitization of mu-opioid receptors (see Infectious_Agents and Related_Receptors). In addition to that, prior exposure of neuronal cells to chemokine treatment enhances the sensitivity of transient receptor potential vanilloid 1 (TRPV1) calcium channel, which is critical for sensing of pain [16204635] (see Sensory/Motor).

Apart from playing an important role in functioning of lymphocytes, chemokines can potentially influence neuronal signaling through the modulation of neuronal calcium currents (see chapter on Chemokine Receptors and Calcium Homeostasis in the Brain). Following interaction with their specific ligands, chemokine receptors trigger a flux in intracellular calcium ions in neurons. This process is sensitive to treatment with pertussis toxin, indicating the involvement of G-protein couple receptors in these mechanisms (see above) [11880151].

In the CNS, activation of neuronal chemokine receptors by their ligands induces calcium transients and activates calcium cAMP-dependent gene transcription factor CREB [9826729] .

Similarly, some chemokines are able to induce a rapid and transient rise in cytosolic free calcium in in either type of T-cell via activation of their ligands [7926371]. It may be of interest to mention here a novel model of reversible inflammatory encephalopathy that is found to be dependent on both genetic and environmental factors. In a study that investigated the pertussis toxin-induced encephalopathy in mice, it was shown that transgenic mice that overexpress MCP-1 manifest transient, severe encephalopathy with high mortality after injections of pertusis toxin, whereas this disorder was significantly milder in mice lacking T-cells. Disruption of CC chemokine receptor 2 (CCR2) abolished both CNS inflammation and encephalopathy in this case [12486156] (see Bacterial lypopolysaccharides)

Several viral proteins that are able to directly interfere with human chemokine receptors have been identified. In the case of the most widely-studied HIV-1, two of its proteins mimick chemokine-mediated calcium signaling and evoke calcium influx through LTCC via activation of several chemokine receptors. One of the chemokine receptors involved, CXCR4, is known for its involvement in immune dysfunction thought to be due to dysregulation of the receptor leading to to enhanced calcium signaling [17169327] (see Infectious_Agents). Through this mechanism it is thought to be able to modulate the migratory and secretory responses of microglia or cause raises in calcium levels in neurons resulting in neuronal injury [10858625, 16553776]. Of particular relevance to autism could be the observed ability of retroviruses to induce neuronal damage even in the absence of lympocyte infiltration, by upregulation of chemokine MCP-1 and subsequent overactivation of it receptor CCR2 [15163738].

Human herpesvirus 6 (HHV-6) encodes highly effective and adaptive chemokine receptor agonists, which will attract CCR2 expressing cells, including macrophages and monocytes. At the same time these proteins enable viral interference in calcium signalling pathways and thus influence many downstream cellular processes. HHV-6 encoded U83A gene is able to induce significant calcium mobilization in cells expressing chemokine receptors CCR1, CCR4, CCR6, or CCR8, with high afinity to both CCR1 and CCR5 on monocytic/macrophage cells. It was concluded that this newly uncovered mechanism could have wider implications for neuroinflammatory diseases such as multiple sclerosis, where both cells bearing CCR1/CCR5 plus their ligands, as well as HHV-6A, have been suggested to play etiological role [16332987, 16365449]. (also see BBB)

Virus-induced immune suppression has been documented with numerous viruses including other members of the herpes virus family, which are able to lower antigen presenting ability of myocites via impaired differentiation of monocytes to dendritic cells, impaired migration of dendritic cells [15914842], loss of up regulation of MHC protein (both Class I and Class II) expression, and loss of up regulation of CD4 cells and CD8 cytotoxic cells, altered cytokine production and activity, altered T cell recognition, and loss of NK function with lack of viral killing [16647570, 1315587, 9087413]. CD4 and CD8 cytotoxic cells and NK cells are affected either directly or indirectly by HHV-6 [9227865, 1348547]. The impairment of immune function by herpes and other viruses may lead to reactivation or emergence of other opportunistic micro-organisms that persist chronically or in a latent state, for example Epstein Bar Virus (EBV), chlamydia, mycoplasma, Borrelia burgdorferi, and babesia, among others. Worth mentioning in this context is the finding that a large subset of subjects with autism shows evidence of various bacterial and/or viral infections not present in age-matched controls (see Infectious_Agents).

It is suggested that one of the mechanisms through which herpesviruses exerts its immunosuppressive effects is via dysregulation of calcium homeostasis in immune cells [14568989]. Another possible example of this mechanism is the effect of HIV-1 viral protein Tat, which is known to inhibit functioning of dendritic cells and NK cells by dysregulating the functioning of LTCC [9516412, 9743356]. In cultured human primary monocytes Tat induces a calcium signal by mobilizing calcium from extracellular stores. This effect is again mediated via calcium influx through LTCC [15661163]. Another viral protein, belonging to Hepatitic C, is known to alter expression of genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, as well as genes regulating immune and inflammatory responses [15349911].

Apart from viruses various other agents are thought to have immunosupressing properties and can act as stressors, negatively influencing proliferative responses of cells. Various toxic chemicals have been shown to induce immune dysregulation via their effects on calcium homeostasis. Cadmium-induced increases in intercellular calcium levels in macrophage cells lead to cellular dysfunction and cell injury, including growth arrest, mitochondrial activity impairment, and necrosis [15254339]. Exposure of cultured murine macrophages to low concentrations of beryllium also induced increases in intracellular calcium and subsequent DNA synthesis. This effect could be reversed by removing extracellular calcium or applying verapamil, indicating the involvement of plasma membrane calcium channels [10380900]. Tin appears to exert similar effects on immune cells via calcium mobilisation [9138773].

Mercurial compounds are particularly toxic to the human immune system. Chronic low-level methylmercury (MeHg) exposure may cause immune disfunction by increasing oxidative stress, affecting transmembrane calcium signaling and splenic cellularity [9653674]. In cultured murine T and B lymphocytes mercury incudes cytotoxicity via several mechanism that involve disturbances in calcium homeostasis and inflammatory cytokine gene expression [12849721]. Adverse affect of methylmercury on the developing immune system through placental and lactational transfer has been demonstrated in rats [1949074]. Intracellular levels of free calcium play an essential role in neutrophil activation, and methylmercury increases those levels in the mouse peritoneal neutrophils via LTCC [12062935]. Exposure of human cultured lymphocytes to methyl mercuric chloride (MeHgCl) results in a sharp decrease in the mitochondrial transmembrane potential in lymphocytes, followed by an increase in oxidative stress [12215206]. Dendritic cells, especially immature dendritic cells, appear to be very sensitive to the effects of organic compound ethylmercurithiosalicylate (thimerosal, primarily present in the tissues as ethylmercury) on calcium homeostasis. Uncoupling of ATP-mediated calcium signaling and dysregulated interleukin-6 secretion has been observed in murine cells following application of relatively small levels thimerosal, with one mechanism involving the uncoupling of ryanodine receptor RyR1 calcium channel complex [16835063]. It should be noted here that in many cells types ryanodine receptors of endoplasmic/sarcoplasmic reticulum form a functionally and structurally connected signalling unit with LTCC on the plasma membrane, and this signaling interaction between LTCC and RyRs is often bi-directional, in that the LTCC trigger the release of intracellular calcium by promoting the opening of nearby RyR (orthograde coupling) while at the same time the activities and function of LTCC can regulated by its interaction with the RyR (retrograde coupling) [11861208].

(see also chapter: Genetic Polymorphisms as Predictors of Inflammatory Outcomes and Chemokine Receptors as Treatment Targets)

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HIV and Autism