Neurotensin is increased in serum of young children with autistic disorder
© Angelidou et al; licensee BioMed Central Ltd. 2010
Received: 9 July 2010
Accepted: 23 August 2010
Published: 23 August 2010
Autism spectrum disorders (ASD) are a group of pervasive neurodevelopmental disorders diagnosed in early childhood. They are associated with a set of "core symptoms" that include disabilities in social interaction skills, verbal and non-verbal communication, as well as repetitive and stereotypic behaviors. There is no definite pathogenetic mechanism or diagnostic tests. Many children with ASD also have "allergic-like" symptoms, but test negative implying mast cell activation by non-allergic triggers. We measured by Milliplex arrays serum levels of 3 neuropeptides that could stimulate mast cells in children with autistic disorder (n = 19; 16 males and 3 females; mean age 3.0 ± 0.4 years) and healthy, unrelated controls (n = 16; 13 males and 3 females; mean age 3 ± 1.2 years). Only neurotensin (NT) was significantly increased from 60.5 ± 6.0 pg/ml in controls to 105.6 ± 12.4 pg/ml in autistic disorder (p = 0.004). There was no statistically significant difference in the serum levels of β-endorphin or substance P (SP). NT could stimulate immune cells, especially mast cells, and/or have direct effects on brain inflammation and ASD.
Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders that include autistic disorder, Asperger's disorder, and pervasive developmental disorder-not otherwise specified (PDD-NOS) [1, 2]. These are diagnosed in early childhood and are characterized by a set of "core symptoms" that include various degrees of disability in social interaction skills, verbal and non-verbal communication, as well as limited interest in activities often associated with repetitive and stereotypic behaviors [1, 2]. The diagnosis of ASD has increased during the last decade to about 1/100 children [3, 4]. However, there is neither distinct pathogenesis, nor reliable biomarkers .
A number of papers have suggested that ASD may be associated with some immune dysfunction in the patient  or the mother during gestation . More recently, evidence has been reviewed suggesting that ASD may have a neuroimmune component involving non-allergic activation of mast cells by triggers such as neuropeptides . However, few studies have measured levels of neuropeptides in autism. Most of the investigations have focused on oxytocin and vasopressin .
In one paper, archived neonatal blood was analyzed with immunoaffinity chromatography and serum levels of vasoactive intestinal peptide (VIP) and calcitonin-gene related peptide (CGRP) were higher in children with ASD (n = 69) and those with mental retardation without ASD (n = 60); in contrast, levels of substance P (SP) and nerve growth factor (NGF) were similar to those of controls . However, a subsequent study by the same author using Luminex immunoaffinity arrays showed no difference in any of these molecules between autistic subjects and controls . Another study showed significantly elevated levels of β-endorphin in the cerebrospinal fluid (CSF) of children with infantile autism (n = 9), but serum levels were not measured .
We investigated serum levels of β-endorphin, neurotensin (NT) and SP, all of which are present both in the brain and the gut and are known mast cell triggers . Here we report that NT is the only neuropeptide significantly elevated in the serum of children with autistic disorder as compared to unrelated age-matched, normally developing, control children.
Patients came from the Second Department of Psychiatry at Attikon General Hospital, University of Athens Medical School (Athens, Greece), an NIH-approved site for biological samples under a collaboration agreement between Athens and Tufts Universities.
All children were assessed by trained ASD clinicians. Parents signed an appropriate consent form according to the Helsinki principles. The ADI-R and ADOS-G scales were used as they have also been validated in the Greek population . The Childhood Autism Rating Scale (CARS) was completed as a further measurement of the severity of ASD. A test for Fragile × syndrome was ordered and the subjects were screened with Wood's Lamp for tuberous sclerosis.
The inclusion and exclusion criteria for the study were as follows:
Must meet ICD-10 criteria for autistic disorder.
Any medical condition likely to be etiological for ASD (e.g. Rett syndrome, focal epilepsy).
Any neurologic disorder involving pathology above the brain stem, other than uncomplicated non-focal epilepsy.
Contemporaneous evidence, or unequivocal retrospective evidence, of probable neonatal brain damage.
Any genetic syndrome involving the CNS, even if the link with autism is uncertain.
Clinically significant visual or auditory impairment, even after correction.
Any circumstances that might possibly account for the picture of autism (e.g. severe nutritional or psychological deprivation).
Active treatment with pharmacological or other agents.
Mastocytosis (including urticaria pigmentosa).
History of upper airway diseases.
History of inflammatory diseases.
History of allergies.
There were no other apparent clinical differences (e.g. gastrointestinal problems) that may have allowed separation of the autistic patients in subgroups. The ASD sample consisted of 19 children with autistic disorder. The controls were normally developing, healthy children, unrelated to the autistic subjects, without any of the exclusion criteria (n = 16; 13 males and 3 females; mean age 3 years); these subjects were seen for routine health visits at the Pediatric Department of the Institute of Social Health Insurance, Greece. There were no identifiers except for age and sex.
Blood was obtained in the morning at least 2 hours after breakfast to minimize any diurnal or postprandial effects. Serum from patients and controls was aliquoted and frozen at -80°C until assayed. All samples were labeled only with a code number, as well as the age and sex of the respective subject. Samples were analyzed for neuropeptides and cytokines using Milliplex MAP, based on the Luminex xMAP technology by Millipore (Billerica, MA).
The results for serum neuropeptide levels are presented as scattergrams, with the horizontal lines indicating the means, in order to appreciate the distribution of the values. The ASD group was compared to the controls using unpaired, unequal, 2-tailed, Student's t-test, as well as the non-parametric Mann-Whitney U test. Significance of comparisons between healthy subjects and subjects with ASD is denoted by p < 0.05.
The ASD group consisted of 19 patients (16 males and 3 females; mean age: 3.0 ± 0.4 years; range: 2.5-3.5 years) all with a diagnosis of autistic disorder. The control group consisted of 16 subjects (13 males and 3 females; mean age: 3 ± 1.2 years; range: 2-5.5 years). These were healthy, normally developing subjects, unrelated to the ASD patients.
We also investigated serum levels of cytokines usually associated with mast cells (IL-4, IL-6, IL-10, IL-13, TNF), but there was no statistically significant difference (results not shown).
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.
This work was funded in part by the National Autism Association, Autism Collaboration and Theta Biomedical Consulting and Development Co., Inc. (Brookline, MA). We thank Dr. Zoe Christoni for help with sample preparation. Asimenia Angelidou and Konstantinos-Dionysios Alysandratos are recipients of scholarships for post-graduate studies from the Hellenic State Scholarships Foundation (Athens, Greece). Bodi Zhang is partially supported by a graduate fellowship from Galenica, SA (Athens, Greece).
- Volkmar FR, State M, Klin A: Autism and autism spectrum disorders: diagnostic issues for the coming decade. J Child Psychol Psychiatry. 2009, 50: 108-115. 10.1111/j.1469-7610.2008.02010.x.View ArticlePubMedGoogle Scholar
- Johnson CP, Myers SM: Identification and evaluation of children with autism spectrum disorders. Pediatrics. 2007, 120: 1183-1215. 10.1542/peds.2007-2361.View ArticlePubMedGoogle Scholar
- Fombonne E: Epidemiology of pervasive developmental disorders. Pediatr Res. 2009, 65: 591-598. 10.1203/PDR.0b013e31819e7203.View ArticlePubMedGoogle Scholar
- Kogan MD, Blumberg SJ, Schieve LA, Boyle CA, Perrin JM, Ghandour RM, et al: Prevalence of parent-reported diagnosis of autism spectrum disorder among children in the US, 2007. Pediatrics. 2009, 5: 1395-1403. 10.1542/peds.2009-1522.View ArticleGoogle Scholar
- Theoharides TC, Doyle R, Francis K, Conti P, Kalogeromitros D: Novel therapeutic targets for autism. Trends Pharmacol Sci. 2008, 29: 375-382. 10.1016/j.tips.2008.06.002.View ArticlePubMedGoogle Scholar
- Ashwood P, Wills S, Van de Water J: The immune response in autism: a new frontier for autism research. J Leukoc Biol. 2006, 80: 1-15. 10.1189/jlb.1205707.View ArticlePubMedGoogle Scholar
- Atladottir HO, Pedersen MG, Thorsen P, Mortensen PB, Deleuran B, Eaton WW, et al: Association of family history of autoimmune diseases and autism spectrum disorders. Pediatrics. 2009, 124: 687-694. 10.1542/peds.2008-2445.View ArticlePubMedGoogle Scholar
- Theoharides TC, Kempuraj D, Redwood L: Autism: an emerging 'neuroimmune disorder' in search of therapy. Exp Opinion on Pharmacotherapy. 2009, 10: 2127-2143. 10.1517/14656560903107789.View ArticleGoogle Scholar
- Heinrichs M, von DB, Domes G: Oxytocin, vasopressin, and human social behavior. Front Neuroendocrinol. 2009, 30: 548-557. 10.1016/j.yfrne.2009.05.005.View ArticlePubMedGoogle Scholar
- Nelson KB, Grether JK, Croen LA, Dambrosia JM, Dickens BF, Jelliffe LL, et al: Neuropeptides and neurotrophins in neonatal blood of children with autism or mental retardation. Ann Neurol. 2001, 49: 597-606. 10.1002/ana.1024.View ArticlePubMedGoogle Scholar
- Nelson PG, Kuddo T, Song EY, Dambrosia JM, Kohler S, Satyanarayana G, et al: Selected neurotrophins, neuropeptides, and cytokines: developmental trajectory and concentrations in neonatal blood of children with autism or Down syndrome. Int J Dev Neurosci. 2006, 24: 73-80. 10.1016/j.ijdevneu.2005.10.003.View ArticlePubMedGoogle Scholar
- Ross DL, Klykylo WM, Hitzemann R: Reduction of elevated CSF beta-endorphin by fenfluramine in infantile autism. Pediatr Neurol. 1987, 3: 83-86. 10.1016/0887-8994(87)90032-4.View ArticlePubMedGoogle Scholar
- Theoharides TC, Kalogeromitros D: The critical role of mast cell in allergy and inflammation. Ann NY Acad Sci. 2006, 1088: 78-99. 10.1196/annals.1366.025.View ArticlePubMedGoogle Scholar
- Papanikolaou K, Paliokosta E, Houliaras G, Vgenopoulou S, Giouroukou E, Pehlivanidis A, et al: Using the Autism Diagnostic Interview-Revised and the Autism Diagnostic Observation Schedule-Generic for the Diagnosis of Autism Spectrum Disorders in a Greek Sample with a Wide Range of Intellectual Abilities. J Autism Dev Disord. 2008, 39: 414-420. 10.1007/s10803-008-0639-6.View ArticlePubMedGoogle Scholar
- Liu J, Zhao J, Di YF, Guo XX, Zhai GR, Huang XH: The dynamic changes of plasma neuropeptide y and neurotensin and their role in regulating cerebral hemodynamics in neonatal hypoxic-ischemic encephalopathy. Am J Perinatol. 2007, 24: 435-440. 10.1055/s-2007-985047.View ArticlePubMedGoogle Scholar
- Bozzola M, Ntodou-Thome A, Bona G, Autelli M, Magnani ML, Radetti G, et al: Possible role of plasma neurotensin on growth hormone regulation in neonates. J Pediatr Endocrinol Metab. 1998, 11: 607-613.PubMedGoogle Scholar
- Carraway R, Leeman SE: The isolation of a new hypotensive peptide, neurotensin, from bovine hypothalami. J Biol Chem. 1973, 248: 6854-6861.PubMedGoogle Scholar
- Castagliuolo I, Leeman SE, Bartolac-Suki E, Nikulasson S, Qiu B, Carraway RE, et al: A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats. Proc Natl Acad Sci USA. 1996, 93: 12611-12615. 10.1073/pnas.93.22.12611.PubMed CentralView ArticlePubMedGoogle Scholar
- Evers BM, Bold RJ, Ehrenfried JA, Li J, Townsend CM, Klimpel GR: Characterization of functional neurotensin receptors on human lymphocytes. Surgery. 1994, 116: 134-139.PubMedGoogle Scholar
- Ramez M, Bagot M, Nikolova M, Boumsell L, Vita N, Chalon P, et al: Functional characterization of neurotensin receptors in human cutaneous T cell lymphoma malignant lymphocytes. J Invest Dermatol. 2001, 117: 687-693. 10.1046/j.0022-202x.2001.01439.x.View ArticlePubMedGoogle Scholar
- Lemaire I: Neurotensin enhances IL-1 production by activated alveolar macrophages. J Immunol. 1988, 140: 2983-2988.PubMedGoogle Scholar
- Carraway R, Cochrane DE, Lansman JB, Leeman SE, Paterson BM, Welch HJ: Neurotensin stimulates exocytotic histamine secretion from rat mast cells and elevates plasma histamine levels. J Physiol. 1982, 323: 403-414.PubMed CentralView ArticlePubMedGoogle Scholar
- Gurney JG, McPheeters ML, Davis MM: Parental report of health conditions and health care use among children with and without autism: National Survey of Children's Health. Arch Pediatr Adolesc Med. 2006, 160: 825-830. 10.1001/archpedi.160.8.825.View ArticlePubMedGoogle Scholar
- Jyonouchi H, Geng L, Cushing-Ruby A, Quraishi H: Impact of innate immunity in a subset of children with autism spectrum disorders: a case control study. J Neuroinflammation. 2008, 5: 52-10.1186/1742-2094-5-52.PubMed CentralView ArticlePubMedGoogle Scholar
- Magalhaes ES, Pinto-Mariz F, Bastos-Pinto S, Pontes AT, Prado EA, Deazevedo LC: Immune allergic response in Asperger syndrome. J Neuroimmunol. 2009, 216: 108-112. 10.1016/j.jneuroim.2009.09.015.View ArticlePubMedGoogle Scholar
- Bakkaloglu B, Anlar B, Anlar FY, Oktem F, Pehlivanturk B, Unal F, et al: Atopic features in early childhood autism. Eur J Paediatr Neurol. 2008, 12: 476-479. 10.1016/j.ejpn.2007.12.008.View ArticlePubMedGoogle Scholar
- Theoharides TC: Autism spectrum disorders and mastocytosis. Int J Immunopathol Pharmacol. 2009, 22: 859-865.PubMedGoogle Scholar
- Akin C, Valent P, Escribano L: Urticaria pigmentosa and mastocytosis: the role of immunophenotyping in diagnosis and determining response to treatment. Curr Allergy Asthma Rep. 2006, 6: 282-288. 10.1007/s11882-006-0061-0.View ArticlePubMedGoogle Scholar
- Castells M: Mast cell mediators in allergic inflammation and mastocytosis. Immunol Allergy Clin North Am. 2006, 26: 465-485. 10.1016/j.iac.2006.05.005.View ArticlePubMedGoogle Scholar
- Kempuraj D, Asadi S, Zhang B, Manola A, Hogan J, Peterson E, et al: Mercury induces inflammatory mediator release from human mast cells. J Neuroinflammation. 2010, 7: 10.1186/1742-2094-7-20.Google Scholar
- Donelan J, Boucher W, Papadopoulou N, Lytinas M, Papaliodis D, Theoharides TC: Corticotropin-releasing hormone induces skin vascular permeability through a neurotensin-dependent process. Proc Natl Acad Sci USA. 2006, 103: 7759-7764. 10.1073/pnas.0602210103.PubMed CentralView ArticlePubMedGoogle Scholar
- Beversdorf DQ, Manning SE, Hillier A, Anderson SL, Nordgren RE, Walters SE, et al: Timing of prenatal stressors and autism. J Autism Dev Disord. 2005, 35: 471-478. 10.1007/s10803-005-5037-8.View ArticlePubMedGoogle Scholar
- Cochrane DE, Carraway RE, Boucher W, Feldberg RS: Rapid degradation of neutotensin by stimulated rat mast cells. Peptides. 1991, 12: 1187-1194. 10.1016/0196-9781(91)90193-S.View ArticlePubMedGoogle Scholar
- Piliponsky AM, Chen CC, Nishimura T, Metz M, Rios EJ, Dobner PR, et al: Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis. Nat Med. 2008, 14: 392-398. 10.1038/nm1738.PubMed CentralView ArticlePubMedGoogle Scholar
- Galli SJ, Kalesnikoff J, Grimbaldeston MA, Piliponsky AM, Williams CM, Tsai M: Mast cells as "tunable" effector and immunoregulatory cells: recent advances. Annu Rev Immunol. 2005, 23: 749-786. 10.1146/annurev.immunol.21.120601.141025.View ArticlePubMedGoogle Scholar
- Theoharides TC, Kempuraj D, Tagen M, Conti P, Kalogeromitros D: Differential release of mast cell mediators and the pathogenesis of inflammation. Immunol Rev. 2007, 217: 65-78. 10.1111/j.1600-065X.2007.00519.x.View ArticlePubMedGoogle Scholar
- Li X, Chauhan A, Sheikh AM, Patil S, Chauhan V, Li XM, et al: Elevated immune response in the brain of autistic patients. J Neuroimmunol. 2009, 207: 111-116. 10.1016/j.jneuroim.2008.12.002.PubMed CentralView ArticlePubMedGoogle Scholar
- Zimmerman AW, Jyonouchi H, Comi AM, Connors SL, Milstien S, Varsou A, et al: Cerebrospinal fluid and serum markers of inflammation in autism. Pediatr Neurol. 2005, 33: 195-201. 10.1016/j.pediatrneurol.2005.03.014.View ArticlePubMedGoogle Scholar
- Croonenberghs J, Bosmans E, Deboutte D, Kenis G, Maes M: Activation of the inflammatory response system in autism. Neuropsychobiology. 2002, 45: 1-6. 10.1159/000048665.View ArticlePubMedGoogle Scholar
- Chez MG, Dowling T, Patel PB, Khanna P, Kominsky M: Elevation of tumor necrosis factor-alpha in cerebrospinal fluid of autistic children. Pediatr Neurol. 2007, 36: 361-365. 10.1016/j.pediatrneurol.2007.01.012.View ArticlePubMedGoogle Scholar
- Gibbs BF, Wierecky J, Welker P, Henz BM, Wolff HH, Grabbe J: Human skin mast cells rapidly release preformed and newly generated TNF-alpha and IL-8 following stimulation with anti-IgE and other secretagogues. Exp Dermatol. 2001, 10: 312-320. 10.1034/j.1600-0625.2001.100503.x.View ArticlePubMedGoogle Scholar
- Cocchiara R, Albeggiani G, Lampiasi N, Bongiovanni A, Azzolina A, Geraci D: Histamine and tumor necrosis factor-a production from purified rat brain mast cells mediated by substance P. Neuroreport. 1999, 10: 575-578. 10.1097/00001756-199902250-00024.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.