Our present results indicate that NT is elevated in the serum of young children with autistic disorder, as compared to unrelated, normally developing controls. All subjects were selected to exclude any allergic, inflammatory or neurologic diseases in order to avoid any confounding factors. The distribution of NT values in the ASD subjects suggests there may be two subgroups. However, nothing in the history or physical examination allowed such subdivision. SP was not elevated as also previously reported [10, 11]; β-endorphin was also not elevated, even though it had been reported to be increased in the CSF of a small group of children (n = 9) with infantile autism .
This is the first study to report measurements of NT in the serum of human subjects with Luminex microbead arrays. No physiological levels of NT have been established for any disease state yet. However, one study reported plasma NT levels measured by RIA in healthy neonates to be 59.1 ± 23.9 pg/ml . Another study evaluated the role of NT in growth regulation in subjects of different ages and reported that plasma levels gradually declined with increasing age with plasma levels in prepubertal children being 22.2 ± 4.8 pg/ml .
NT is a peptide originally isolated from the brain . It is present also in the gastrointestinal tract, where it can induce intestinal inflammation . NT can stimulate lymphocyte proliferation , activate T cells , and enhance IL-1 production from macrophages . NT is also a potent trigger of mast cells . These effects are relevant to the findings that many children with ASD also present with gastrointestinal and "allergic-like" symptoms . In particular, mast cells appear to be activated in ASD as suggested by more food allergies  and increased atopic symptoms in Asperger patients . Such "allergic symptoms" often occur in the absence of elevated serum IgE or positive skin prick tests , suggesting mast cell activation by non-immune triggers . Moreover, a preliminary report indicated that ASD is 10-times more frequent in mastocytosis patients (1/10 children)  than the general population (1/100 children) . Mastocytosis is a rare disorder, which presents with skin reactions, food allergies or food intolerance, diarrhea, anxiety , but also lack of concentration ("brain fog"), and hyperactivity .
NT could be released from the brain, the intestines or dorsal root ganglia and could act together with environmental triggers such as mercury  or corticotropin-releasing hormone (CRH), secreted under stress, to stimulate mast cells and lead to neurogenic inflammation . The possible release of NT under stress may be relevant to the finding of higher incidence of prenatal stressors in mothers of children with ASD . The present study did not attempt to correlate serum NT to levels of stress. Future studies could employ the Hamilton Anxiety Scale or the Spielberger's State-Trait-Anxiety Inventory, as well as measure serum levels of cortisol, adrenocorticotropic hormone (ACTH) and CRH. It is interesting that mast cells can degrade NT [33, 34]. This action suggests that mast cells have developed a rapid mechanism for limiting stimulation by NT that implies an important pathophysiological role.
Mast cells are involved in both innate and acquired immunity , as well as in inflammation . Moreover, mast cells can release some mediators "selectively", without concomitant secretion of either one of their "flagship" molecules histamine or tryptase , making serum measurement of these mediators as biomarkers irrelevant. Moreover, histamine is metabolized rapidly, while tryptase is elevated only in anaphylaxis and mastocytosis, conditions that were part of the exclusion criteria. It is not apparent at present which mast cell mediators are secreted in response to NT. Our preliminary findings on Luminex measurements of IL-4, IL-5, IL-6, IL-8, IL-13 and tumor necrosis factor (TNF) did not show any significant difference (results not shown). Even though IL-6 expression was elevated in the brains of deceased ASD patients , and it was detected at low levels in the CSF in subjects with autism (n = 12), it was not significantly elevated in the serum of autistic subjects (n = 35) compared to control subjects with other neurologic disorders (n = 21) . Elsewhere, there was only a trend towards increased production of IL-6 and TNF-α in whole blood of autistic children as compared to normal controls . Another study reported that TNF-α levels in CSF of patients with ASD (n = 10) were significantly higher than concurrent serum levels . TNF is uniquely stored in mast cell granules , and brain mast cells were reported to secrete TNF . It may well be that NT levels are sufficient to activate only brain and gut mast cells, thus not significantly raising systemic levels. Alternatively, mast cells may release additional mediators that have not been identified so far.
The present results indicate that NT is increased in young children with autistic disorder and could participate in altered innate immunity and brain inflammation.