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Problem: There is a need to collect the many and varied data on AD/HD (Attention deficit hyper-activity disorder) into a meaningful overview. Method: Based on peer reviewed and published data as well as own research we try to make sense of the physiological mechanisms resulting in the relevant symptoms. Conclusion: AD/HD clearly has a genetic disposition, but as with many other genetic syndromes, the resulting proteomics must be stressed to become manifest as disease/ disorder. A common trait in the different etiologies is lower arousal.

Although mutations in the MECP2 gene are associated with many cases of Rett syndrome (RS), the phenotype remains unexplained. At least in the early stages, RS and autism have common features; urinary analysis of autistic children has shown an abnormal profile of excretion of sulphur-containing anions, with high levels of sulphite, sulphate and thiosulphate and low values for thiocyanate ions. These parameters were therefore studied in an RS population. Levels of urinary peptides, protein, cysteine, free sulphate, conjugated sulphate, sulphite, thiosulphate and thiocyanate were determined in female RS patients ( n = 21) and in controls ( n = 21), using standard methods. The urinary volumes were adjusted to standard creatinine levels (200 nmol/mL). Children with RS had higher urinary peptides and protein excretion. Free sulphate values and total sulphate excretion were higher in RS ( P P P

Problem: Some researchers have not found the opioids in urine of autistic children. We have therefore looked at this problem again. Method: Mass spectrometry and fragmentation mass spectrometry (MS/MS) have been carried out on peaks from the HPLC that show co-chromatography with synthetic standards and peaks that are shared by different autistic children. Results: In quickly frozen urine we find the presence of exorphins, and can also demonstrate a rather fast break down at room temperature of these peptides in urine. Conclusion: Exorphins are present in urine in autistic children, but must be protected against break down and aggregation by fast freezing or acetic acid and adjusting declustering potential and collision potential during mass-spectroscopy. Specific antibody increases and the effect of removing precursor proteins from the diet reinforce this view.

Exorphins from casein and gluten have been found by HPLC and mass spectroscopy with fragmentation pattern in quickly frozen urine. Removing the proteins that contain these peptides, by dietary intervention has been tried with behavioral effects. We wanted to know how fast such changes take place. Method: Parents and caregivers filled out ATEC scores (Autism treatment evaluation checklist) over time so that changes in scores could be registered. Results: In this group of children who all responded to diet the time required for a positive effect was months rather than weeks. Conclusion: Short term interventions are probably a waste of time and money, and at least 3 - 6 months trials seem to be necessary.

Background: Autoimmune phenotypes are prevalent in major psychiatric disorders. Disequilibria of cellular processes occurring in the gastrointestinal (GI) tract likely contribute to immune dysfunction in psychiatric disorders. As the venue of a complex community of resident microbes, the gut in a homeostatic state equates with a functional digestive system, cellular barrier stability and properly regulated recognition of self and non-self antigens. When gut processes become disrupted as a result of environmental or genetic factors, autoimmunity may ensue. Methods: Here, we review the issues pertinent to autoimmunity and the microbiome in psychiatric disorders and show that many of the reported immune risk factors for the development of these brain disorders are in fact related and consistent with dysfunctions occurring in the gut. We review the few human microbiome studies that have been done in people with psychiatric disorders and supplement this information with mechanistic data gleaned from experimental rodent studies. Results: These investigations demonstrate changes in behavior and brain biochemistry directly attributable to alterations in the gut microbiome. We present a model by which autoantigens are produced by extrinsicallyderived food and microbial factors bound to intrinsic components of the gut including receptors present in the enteric nervous system. Conclusion: This new focus on examining activities outside of the CNS for relevance to the etiology and pathophysiology of psychiatric disorders may require new modalities or a re-evaluation of pharmaceutical targets found in peripheral systems.

Problem: The nature of the peptides found increased in urine from autism needs verification of their structure, especially those that show opioid activity. Methods: The peptides were separated on reverse phase C-18 HPLC in Trifluoroacetic acid–acetonitril gradients. Peaks eluting where synthetic opioids appear, and peaks that are common to most autistic children were analyzed by mass spectrometry and fragmentation pattern on a quadropole mass-spectrometer. Results: We could demonstrate exorphins in the urine from autistic children, and their length varied from one patient to the next. Conclusion: Exorphins are found in urine of autistic children and may account for their symptoms.

Hyperpeptiduria and opioid excess have been reported in schizophrenia. According to Prof. Dr. L. Lindstrom, Sweden opioids may explain the patho-physiology of this syndrome. Therefore it is critical to elucidate the presence and nature of opioids in schizophrenia and diagnostic sub groups. First morning urine from untreated schizoaffective patients (ICD-10: F 25.1) was separated on HPLC and peaks that elute where different opioid standards appear, freeze dried, re-dissolved in methanol/water (50/50) and 10mM formic acid. Mass spectrometry and MS/MS or fragmentation mass spectrometry was performed. We found fragmentation pattern of beta-casomorphin 1-3 and 1-4 (bovine) identical to synthetic standards from Bachem. The aggregation tendency of peptides was much in evidence. The reported exorphins were found in the urine from 8 of 12 untreated schizoaffective patients.

Biochemical, immunological and epidemiological evidence increasingly support the suggestion that there is a causal relationship between gluten/gliadin and schizophrenia as originally proposed by F. C. Dohan. Furthermore the necessary physiological mechanisms exist to explain a mechanism involving bioactive peptides from these proteins, and these show that this mechanism is possible and probable in at least in a substantial subgroup of schizophrenic patients. Evidence shows a fairly strong genetic disposition, and it must be recognised that any genetic mechanism must implicate altered chemistry and function of proteins. Evidence supports the likelihood that dietary intervention is beneficial for some, and this demands further investigation. A similar conclusion may apply to autism spectrum conditions.

The integrin-β 3 gene (ITGB3), located on human chromosome 17q21.3, was previously identified as a quantitative trait locus (QTL) for 5-HT blood levels and has been implicated as a candidate gene for autism spectrum disorder (ASD). We performed a family-based association study in 281 simplex and 12 multiplex Caucasian families. ITGB3 haplotypes are significantly associated with autism (HBAT, global P=0.038). Haplotype H3 is largely over-transmitted to the affected offspring and doubles the risk of an ASD diagnosis (HBAT P=0.005; odds ratio (OR)=2.000), at the expense of haplotype H1, which is under-transmitted (HBAT P=0.018; OR=0.725). These two common haplotypes differ only at rs12603582 located in intron 11, which reaches a P-value of 0.072 in single-marker FBAT analyses. Interestingly, rs12603582 is strongly associated with pre-term delivery in our ASD patients (P=0.008). On the other hand, it is SNP rs2317385, located at the 5' end of the gene, that significantly affects 5-HT blood levels (Mann-Whitney U-test, P=0.001; multiple regression analysis, P=0.010). No gene-gene interaction between ITGB3 and SLC6A4 has been detected. In conclusion, we identify a significant association between a common ITGB3 haplotype and ASD. Distinct markers, located toward the 5' and 3' ends of the gene, seemingly modulate 5-HT blood levels and autism liability, respectively. Our results also raise interest into ITGB3 influences on feto-maternal immune interactions in autism.

There is increasing interest in the use of gluten- and casein-free diets for children with autism spectrum disorders (ASDs). We report results from a two-stage, 24-month, randomised, controlled trial incorporating an adaptive 'catch-up' design and interim analysis. Stage 1 of the trial saw 72 Danish children (aged 4 years to 10 years 11 months) assigned to diet (A) or non-diet (B) groups by stratified randomisation. Autism Diagnostic Observation Schedule (ADOS) and the Gilliam Autism Rating Scale (GARS) were used to assess core autism behaviours, Vineland Adaptive Behaviour Scales (VABS) to ascertain developmental level, and Attention-Deficit Hyperactivity Disorder - IV scale (ADHD-IV) to determine inattention and hyperactivity. Participants were tested at baseline, 8, and 12 months. Based on per protocol repeated measures analysis, data for 26 diet children and 29 controls were available at 12 months. At this point, there was a significant improvement to mean diet group scores (time*treatment interaction) on sub-domains of ADOS, GARS and ADHD-IV measures. Surpassing of predefined statistical thresholds as evidence of improvement in group A at 12 months sanctioned the re-assignment of group B participants to active dietary treatment. Stage 2 data for 18 group A and 17 group B participants were available at 24 months. Multiple scenario analysis based on inter- and intra-group comparisons showed some evidence of sustained clinical group improvements although possibly indicative of a plateau effect for intervention. Our results suggest that dietary intervention may positively affect developmental outcome for some children diagnosed with ASD. In the absence of a placebo condition to the current investigation, we are, however, unable to disqualify potential effects derived from intervention outside of dietary changes. Further studies are required to ascertain potential best- and non-responders to intervention. The study was registered with ClincialTrials.gov, number NCT00614198.

Problem behaviour hampers learning and the normal development of skills and competencies. The children in focus in this article from Norway are six-year-olds with persistent problem behaviour. Early identification of these children is imperative for the implementation of structured educational interventions. Knowledge about their behaviour and skills is therefore important. The aim of the study reported here by Ann-Mari Knivsberg, professor of special education at the National Centre for Reading Education and Research at the University of Stavanger, Synnove Iversen, physiotherapist and research fellow at the University of Bergen, Magne Nodland, speech therapist and special educator, and Karl-L Reichelt, senior consultant and researcher at the Paediatric Research Institute at the National Hospital in Oslo, was to assess the participants' behaviour and their cognitive, linguistic and motor skills and to correlate behaviour and skills. The participants were 31 children enrolled in a ‘high-risk’ programme for children with problem behaviour. Standardised test materials were used to obtain information on their behaviours and skills. Social problems, attention problems, anxiety and depressive traits were the most frequently reported behavioural problems. Linguistic delay, lower than normal cognitive scores and severe motor co-ordination difficulties were also detected. Correlations were found between various behavioural traits and between the behaviour and the skills. Attention problems were most frequently correlated to cognitive, linguistic and motor functioning. At the end of their article, the authors call for multi-disciplinary approaches to the design of future assessment and intervention strategies and to further research into the effectiveness of available screening instruments and intervention programmes.

Autistic syndromes are characterized by impaired social, communicative, and imaginative skills. Urinary peptide abnormalities, in part due to gluten and casein, have been detected in some individuals With autism. These abnormalities reflect processes With opioid effect, Which may explain the behavioral abnormalities seen in autism. The aim of this single-blind, controlled study Was to evaluate the effect of a gluten-free and casein-free diet for children With autism and urinary peptide abnormalities. Observations and tests Were carried out With the 20 participanting children before they Were randomly assigned to either the diet or the control group. The experimental period Was 1 year, after Which observations and tests Were repeated. Significant reduction of autistic behavior Was registered for participants in the diet group, but not for those in the control group.

Impaired social interaction, communication and imaginative skills characterize autistic syndromes. In these syndromes urinary peptide abnormalities, derived from gluten, gliadin, and casein, are reported. They reflect processes with opioid effect. The aim of this single blind study was to evaluate effect of gluten and casein-free diet for children with autistic syndromes and urinary peptide abnormalities. A randomly selected diet and control group with 10 children in each group participated. Observations and tests were done before and after a period of 1 year. The development for the group of children on diet was significantly better than for the controls.

The melanocyte-inhibiting tripeptide (MTP) pyroGlu-Phe-GlyNH2 is present in tissue cultures of non-transformed melanocytes and melanoma cells and influences melanocyte growth in vitro. The objective of the present study was to investigate a possible effect of MTP on the in vivo growth of B16A2, a monoclonal experimental melanoma. The B16A2 clone was established by the limited dilution technique. It has a reduced DNA content and displays slower growth both in vivo and in vitro compared to the parent cell line (B16). B16A2 cells were injected subcutaneously into hairless mice at four sites (300 000 cells in 0.25 ml buffer/site). MTP was given by i.p. injection 3 times a week at two concentrations (1 pmol and 1 nmol/animal). The control animals received the equal volume of solvent. The animals were sacrificed 1 and 2 weeks after tumour transplantation, and all tumours were weighed. One week after transplantation, the animals who received 1 pmol MTP had fewer tumours and a reduced tumour load. Two weeks after the transplantation, the differences between control and treated animals were no longer observed. The results indicate that MTP temporarily delays in vivo tumour growth.

Anni-Mari Knivsberg, Karl-L. Reichelt and Magne Nodland outline the problems resulting from comorbidity (the simultaneous occurrence of two or more disorders) in children, the effects of which tend to be more serious than those resulting from a single disorder. Two of the most common developmental disorders are dyslexia and ADHD (attention deficit hyperactivity disorder), which frequently co-occur and affect almost all developmental areas. It is, therefore, important to identify comorbidity before planning and implementing educational programs. In considering the implications of comorbidity, a comprehensive review of the international literature is provided.

Abnormal urinary peptide patterns and peptide levels are reported in autism. These abnormalities are in part peptides from gluten, gliadin and casein, and some of them are opioids. These opioids cross the blood-brain barrier and effect the central nervous system. A seven year old girl with autistic behaviour who benefited from a diet without gluten and casein is described in this case report. The young girl's behaviour and skills were registered before diet, after one year and after two. Over the period of two years the girl changed from being severely withdrawn to a normally communicating child who enjoys the company of others. The child's linguistic and cognitive skills also increased simultaneously more than could be expected. Furthermore her urine patterns and peptide levels were normalised.

Cell growth and differentiation in melanocyte cell populations are regulated by a wide range of bioactive substances. Recently, the tripeptide pyroGlu-Phe-GlyNH2 which inhibits melanocyte growth in vitro was identified in both murine nontransformed melanocytes and malignant melanoma cells. The present study was undertaken to investigate the cell cycle specificity as well as the growth inhibitory profile of the tripeptide after a single or repeated administration to melanocyte cultures. Dose-related effects of the peptide were studied using three different bioassay systems: estimation of cell number, DNA synthesis, and cell flux into mitosis. Growth of melanocyte cultures as well as melanocyte mitotic activity were found to be reduced significantly by the tripeptide at two separate dose levels (10(-11) and 10(-14)-10(-15) M). Growth inhibition of melanocyte population did not last long: less than 36 h after the first and less than 24 h after the second peptide addition to the cultures. The level of DNA synthesis in melanocytes remained unchanged after a single peptide administration. The findings indicate that the tripeptide pyroGlu-Phe-GlyNH2 causes transitory delay of cell growth in cultured melanocyte population resulting from a reversible inhibition of melanocyte transition from the G2-phase of the cell cycle into mitosis.

L'activation du reflexe humoral larynge provoquant un reflux gastro-oesophagien, peut constituer un mecanisme pathogenique de l'apnee en liaison avec un risque pour la vie et le SIDS. Les enfants souffrant de reflux gastro-oesophagique et d'apnee presentent un taux accru d'immuno-reactivite de la β-endorphine dans le plasma. La β-endorphine peut inhiber la respiration et induire la bradycardie. La naloxone est un antagoniste de la β-endorphine et il a ete etabli qu'elle peut etre utilisee avec succes pour soigner les enfants souffrant d'apnee et presentant un niveau accru d'immuno-reactivite de β-endorphine du liquide cephalo-rachidien. Dans le but de determiner si la β-endorphine du liquide cephalo-rachidien augmentait avec le reflexe humoral larynge induisant l'apnee chez le porcelet, nous avons provoque un reflexe humoral larynge avec et sans pre-traitement a la naloxone et nous avons mesure l'immuno-reactivite de la β-endorphine dans le liquide cephalo-rachidien avant et apres l'episode d'apnee. Nous avons pris des porcelets de t a 10 jours qui avaient suivi une premedication a l'azaperone et auxquels nous avons administre de la metomidate jusqu'a etablissement d'une anesthesie adequate. Les porcelets ont subi une ventilation mecanique jusqu'a 60 min apres l'intervention, apres quoi ils respiraient spontanement. Nous avons enregistre le rythme respiratoire, le volume minute et le volume courant. Pendant l'intervention, nous avons place une sonde vesicale dans l'espace subglottal pour insuffler le larynx avec l'air sature d'ammoniaque destine a stimuler le reflexe humoral larynge. Nous avons aussi place un catheter arteriel pour enregistrer la pression sanguine. La pouls etait enregistre par electrodes sur la peau. Nous avons administre a un groupe de porcelets 100 microgrammes/kg de naloxone i. v. 5 minutes avant le reflexe humoral larynge induisant l'apnee (n = 6) tandis que l'autre groupe n'etait pas traite (n = 7). Le liquide cephalo-rachidien a ete preleve avant et apres chaque episode d'apnee par ponction sous-occipitale. Les taux de β-endorphine ont ete mesures par dosage immuno-radiologique du liquide cephalo-rachidien. Les surnageants de l'examen immuno-radiologique ont ete transferes a une HPLC pour verifier que la β-endorphine avait ete mesuree. Nous avons applique le test Mann-Whitney U pour comparer les deux groupes et, pour les examens de correlation, nous nous sommes servi du coefficient de correlation des rangs de Spearman. Nous avons constate que la duree moyenne des episodes e'apnee chez les porcelets traites a la naloxone etait nettement plus courte que chez les porcelets non traites (n = 0,02) avec respectivement 12 sec (plage de 0 a 20) et de 28 sec (plage de 10 a 40). Pour le reste, l'accroissement de la pression sanguine et le ralentissement du rythme cardiaque pendant le reflexe humoral larynge provoquant l'apnee chez les porcelets traites a la naloxone ne presentait aucune difference significative par rapport aux porcelets non traites. Le niveau d'immuno-reactivite de la β-endorphine du liquide cephalo-rachidien avant l'apnee etait inferieur a 4,3 fmol/ml SCF. Le niveau moyen d'immuno-reactivite de la β-endorphine du liquide cephalo-rachidien apres l'apnee les porcelets traites a la naloxone ne presentait pas de difference statistiquement pertinente par rapport aux porcelets non traites avec respectivement 615 fmol/ ml CSF (plage de 93 a 1090) et 984 fmol/ml CSF (plage de 380 a 2400) (figure 1). Apres transfert du materiel d'immuno-reactivite de la β-endorphine du dosage immuno-radiologique a la HPLC, nous avons du demontrer l'identite avec la )-endorphine ajoutee. Nous avons aussi mis en relation le niveau d'immuno-r

Dietary intervention as a tool for maintaining and improving physical health and wellbeing is a widely researched and discussed topic. Speculation that diet may similarly affect mental health and wellbeing particularly in cases of psychiatric and behavioral symptomatology opens up various avenues for potentially improving quality of life. We examine evidence suggestive that a gluten-free (GF), casein-free (CF), or gluten- and casein-free diet (GFCF) can ameliorate core and peripheral symptoms and improve developmental outcome in some cases of autism spectrum conditions. Although not wholly affirmative, the majority of published studies indicate statistically significant positive changes to symptom presentation following dietary intervention. In particular, changes to areas of communication, attention, and hyperactivity are detailed, despite the presence of various methodological shortcomings. Specific characteristics of best- and non-responders to intervention have not been fully elucidated; neither has the precise mode of action for any universal effect outside of known individual cases of food-related co-morbidity. With the publication of controlled medium- and long-term group studies of a gluten- and casein-free diet alongside more consolidated biological findings potentially linked to intervention, the appearance of a possible diet-related autism phenotype seems to be emerging supportive of a positive dietary effect in some cases. Further debate on whether such dietary intervention should form part of best practice guidelines for autism spectrum conditions (ASCs) and onward representative of an autism dietary-sensitive enteropathy is warranted.

To determine whether β-endorphin is involved in the laryngeal chemoreflex. we initially injected 0.01 1 mg of β-endorphin into the cisterna magna (i.c.m.) and registered the respiralory and cardiovascular patterns in 5–10-d-old piglets. From 0.1 to 1 mg of β-cndorphin i.c.m. induced a decrease in the minute volume, hearl rate, and blood pressure within 15 min. Within the next 30 min respiratory pauses accompanied by blood pressure increases and reductions in heart ratc developed, similar to the respiratory and cardiovascular pattern of the induced laryngcal chemoreflex. Based on these initial data, we decided to induce a laryngeal chemoreflex in piglets pretreated with 0.1 mg of β-endorphin i.c.m (n = 6), 0.2 mg of β-endorphin i.c.m. (n = 6), 0.1 mg of β-endorphin i.c.m. and 100 μg/kg naloxone i.v. (n = 6), 100 μg/kg naloxonc i.v. (n - 6). or water i.c.m. (n = 6). Because clevated levels of hypoxanthine in the vitreous humor may indicate hypoxia before death, we therefore measured the postmortem hypoxanthine levels in the vitreous humor. The laryngeal chemoreflex-induced apnea was shortenec in the piglet group pretreated with water i.c.m and naloxone i.v. (p < 0.01) and in the piglet group pictreated with 0.1 mg of β-endorphin i.c.m and naloxone i.v. (p < 0.05), but not significantly prolonged in the piglet groups pretreated with 0.1 or 0.2 mg of β-endorphin i.c.m. when compared with the piglcts prerreated with water i.c.m. The hypoxanthine levels in the vitreous humor were found increased after death only in piglets prctreated with 0.2 mg of β-endorphin i.c.m. before the laryngeal chemorefltex was induced (p < 0.05). We conclude that β-cndorphin is probably a mediator of apnca induced by the laryngenl chemoreflex in piglets.

IgA antibody levels in serum were examined in two groups of schizophrenic patients. All were diagnosed according to DSM-III-R criteria. One group of 36 males and 12 females were compared to historical controls. The other group consisted of 13 males off drugs for at least 3 months; these were compared with age- and sex-matched controls. An increase in specific IgA antibodies was found. More schizophrenics than controls showed IgA antibody levels above the upper normal limit to gliadin, beta-lactoglobulin, and casein.

A single intraperitoneal (i.p.) injection of picomolar doses of the epidermal pentapeptide (EPP), pGlu-Glu-Asp-Ser-GlyOH, is followed by a reversible inhibition of mouse epidermal cell proliferation. An equimolar mixture of zinc and EPP injected i.p. into hairless mice reversed the inhibitory activity, resulting in an immediate stimulation of epidermal G2-M cell flux. The stimulatory effect was strongest at the lowest dose (5 pmol). This effect was probably caused by a dizinc-dipentapeptide dimer, as shown by gel filtration and atomic emission spectrometry. When zinc was added in excess (EPP:Zn 1:9) no such dimer could be identified, and the mixture had no stimulatory effect. The results are discussed in terms of epidermal cell kinetics, and in relation to the use of zinc in dermatology.

Problem: The nature of the peptides found increased in urine from autism needs verification of their structure, especially those that show opioid activity. Methods: The peptides were separated on reverse phase C-18 HPLC in Trifluoroacetic acidacetonitril gradients. Peaks eluting where synthetic opioids appear, and peaks that are common to most autistic children were analyzed by mass spectrometry and fragmentation pattern on a quadropole mass-spectrometer. Results: We could demonstrate exorphins in the urine from autistic children, and their length varied from one patient to the next. Conclusion: Exorphins are found in urine of autistic children and may account for their symptoms. Keywords: autism; peptides; exorphins; mass-spectroscopy (Published: 24 August 2012) Citation: Microbial Ecology in Health & Disease 2012, 23 : 18958 - http://dx.doi.org/10.3402/mehd.v23i0.18958

An increased level of immunoglobulin A (IgA) antibodies to food antigens has been found in Down syndrome. Biopsy did not show any anomaly of the gut. Increased IgA levels against specific proteins reflect increased transmucosal transport of antigens. The antigens presented may be immunogenic peptides or intact antigens. In this paper, we wished to exclude an institutional cause such as infections and also age differences.

In nucleus tractus solitarius (NTS) beta-endorphin (BEND) induces bradycardia and respiratory depression which have been reported to precede death in sudden infant death (SID). Of SID victims, 50% have elevated levels of beta-endorphin immunoreactivity (BENDI) in the cerebrospinal fluid (CSF), and 50% had undetectable levels. We therefore investigated the relationship of BENDI in the CSF to BENDI levels in the NTS area. This study included SID victims (CSF from n = 47, brain stem from n = 16), borderline SID victims (CSF and brain stem from n = 2), sudden death in childhood (CSF and brain stem from n = 1), and controls (CSF from n = 32, brain stem from n = 11). BEND in CSF and NTS area, after extraction, was measured by radioimmunoassay. High performance liquid chromatography was used for closer identification of BENDI. We found that the SID victims divided into two subpopulations, one having a relatively high BENDI level in CSF and one having no detectable level (P < 0.01). Furthermore, an inverse relationship was found between BENDI level in CSF and BENDI level in NTS area in the SID victims (P < 0.05). We conclude that increased BENDI level in CSF is associated with low BENDI level in the NTS area in 50% of SID victims. The low BENDI level in the NTS area may be due to increased release of BEND.

Beta-endorphin (BEND) may induce respiratory depression. Elevated levels of beta-endorphin immunoreactivity (BENDI) in the CSF are found in children with apnoea and in about 50% of sudden infant death (SID) victims. Premortal hypoxia in SID victims has been indicated by elevated hypoxanthine (HX) levels in the vitreous humour (VH). In this study we correlated BENDI in CSF with HX in VH in SID victims (n=19) and controls (n=18). BEND in CSF was measured by RIA, and HPLC was used for identification of BENDI. HX in VH was measured by HPLC. All the SID victims had elevated levels of HX in VH. The BENDI in CSF divided the SID victims into two subpopulations (P

Autism is a complex neurodevelopmental disorder, likely encompassing multiple pathogenetic components. The aim of this study is to begin identifying at least some of these components and to assess their association with biological endophenotypes. To address this issue, we recruited 245 Italian patients with idiopathic autism spectrum disorders and their first-degree relatives. Using a stepwise approach, patient and family history variables were analyzed using principal component analysis ("exploratory phase"), followed by intra- and inter-component cross-correlation analyses ("follow-up phase"), and by testing for association between each component and biological endophenotypes, namely head circumference, serotonin blood levels, and global urinary peptide excretion rates ("biological correlation phase"). Four independent components were identified, namely "circadian & sensory dysfunction," "immune dysfunction," "neurodevelopmental delay," and "stereotypic behavior," together representing 74.5% of phenotypic variance in our sample. Marker variables in the latter three components are positively associated with macrocephaly, global peptiduria, and serotonin blood levels, respectively. These four components point toward at least four processes associated with autism, namely (I) a disruption of the circadian cycle associated with behavioral and sensory abnormalities, (II) dysreactive immune processes, surprisingly linked both to prenatal obstetric complications and to excessive postnatal body growth rates, (III) a generalized developmental delay, and (IV) an abnormal neural circuitry underlying stereotypies and early social behaviors.

A test system for growth regulators based on the time course of liver regeneration in male NMRI mice injected intraperitoneally (ip) with 50 nmol CCl4 at 12 is described. Regenerative DNA synthesis (labelling index) peaked at 36 h after CCl4 injury, and the Colcemid-assessed mitotic rate (MR) at 42 h, i.e., 6 h later. This response pattern was used to assess the effects of factors in liver extracts that regulate or modulate hepatocyte proliferation. The effect of one, two, four or eight ip injections of an aqueous mouse liver extract on MR was tested at 48 h. A 30-70% inhibition was seen only after single injections at 12 h, 29 h or 44 h after CCl4 treatment. A 30-80% stimulation was observed after a single injection of the liver extract at 0, 5 or 24 h, and after two or four injections. The assay system could thus detect the presence of growth modulators in the extract. The experiments also showed that the timing was crucial. We recently isolated and characterized a growth inhibitory pentapeptide from mouse liver extracts. Using a synthetic pentapeptide with the same structure we reassessed the timing for growth inhibition seen with the liver extract. The following test system for growth inhibitors seemed most expedient: inhibitor administration at 29 h to affect G1-S transition, measured as reduced DNA synthesis at 36 h, or inhibitor administration at 44 h to affect G2-M transition, measured as reduced MR at 48 h.

The subcellular distribution of gamma-glutamylamino-transferase (EC 2.3.2.2) and transglutaminase (EC 2.3.2.1) has been investigated in rat brain tissue fractionated by a centrifugation and sedimentation technique. Neither of these enzymes was enriched in the synaptosomal fraction. Comparing the in vitro grown astrocytes with synaptosomes, we find that both of these enzymes may possibly be more important in the glial element of the synaptic region. gamma-Glutamylaminotransferase is most abundant in capillaries, confirming previous reports.

The peptide pyroGlu-Gln-Gly-Ser-Asn, recently isolated from mouse liver, inhibited DNA synthesis and proliferation in vitro of MH1C1 cells, a rat clonal strain derived from a Morris transplantable hepatoma. Both the biological peptide isolated from mouse liver and the synthetic homolog showed bell-shaped dose-response curves. Maximal inhibition (approximately 50%) was observed at two separate dose ranges: one at 10(-7)-10(-10) M, and one at 10(-14)-10(-15) M.

Autism is a complex and life-long behavioural disorder of unknown aetiology. Recent reports have indicated the involvement of digestive tract dysfunction and possible complications from inadequate nutrition. In this study, 34 autistic children (12 untreated and 22 receiving therapeutic treatments related to digestive function and nutritional uptake) and 29 control subjects (all 5-15 years of age) were investigated to determine whether there were any anomalies in the urinary excretion of amino acids, glucose, sucrose, arabinose and tartaric acid using GC/FID and GC/MS analysis techniques. Significantly lower relative urinary levels of essential amino acids were revealed for both the untreated (mean +/- SEM, 32.53 +/- 3.09%) and treated (31.98 +/- 2.87%) autistic children compared with the controls (37.87 +/- 1.50%). There were no significant differences in measured excretions of sugars or tartaric acid. It was concluded that the untreated autistic children had evidence of altered metabolic homeostasis.

Protein kinase C enzymes play an important role in signal transduction, regulation of gene expression and control of cell division and differentiation. The fsI and betaII isoenzymes result from the alternative splicing of the PKCbeta gene (PRKCB1), previously found to be associated with autism. We performed a family-based association study in 229 simplex and 5 multiplex families, and a postmortem study of PRKCB1 gene expression in temporocortical gray matter (BA41/42) of 11 autistic patients and controls. PRKCB1 gene haplotypes are significantly associated with autism (P

Earlier work has shown that epidermal cells contain a peptide, pyroGlu-Glu-Asp-Ser-GlyOH, that induces a moderate but long-lasting inhibition of epidermal cell proliferation when given at low (picomol) doses ip in vivo and in vitro. In the present study, the epidermal pentapeptide was applied topically to the back skin of hairless mice at different concentrations and in a water-miscible cream. A single topical application of either high (0.25% wt/wt) or low (0.004% or 0.02% wt/wt) doses of the pentapeptide was followed by oscillations in epidermal DNA synthesis and G2-M cell flux (mitotic rate). In general, epidermal cell proliferation was inhibited during the first 10-day period after treatment with the two lower doses, while the highest concentration of pentapeptide (0.25%) stimulated epidermal cell proliferation. In spite of the effects on epidermal cell proliferation the size of the epidermal cell population in the treated area (number of nucleated cells and epidermal thickness) showed no corresponding alterations. The results could imply that the epidermal pentapeptide modifies epidermal cell proliferation and terminal differentiation in such a way that the two are balance with each other.

Head growth rates are often accelerated in autism. This study is aimed at defining the clinical, morphological, and biochemical correlates of head circumference in autistic patients.Fronto-occipital head circumference was measured in 241 nonsyndromic autistic patients, 3 to 16 years old, diagnosed according to DSM-IV criteria. We assessed 1) clinical parameters using the Autism Diagnostic Observation Schedule, Autism Diagnostic Interview-Revised, Vineland Adaptive Behavioral Scales, intelligence quotient measures, and an ad hoc clinical history questionnaire; 2) height and weight; 3) serotonin (5-HT) blood levels and peptiduria.The distribution of cranial circumference is significantly skewed toward larger head sizes (p.00001). Macrocephaly (i.e., head circumference97th percentile) is generally part of a broader macrosomic endophenotype, characterized by highly significant correlations between head circumference, weight, and height (p.001). A head circumference75th percentile is associated with more impaired adaptive behaviors and with less impairment in IQ measures and motor and verbal language development. Surprisingly, larger head sizes are significantly associated with a positive history of allergic/immune disorders both in the patient and in his/her first-degree relatives.Our study demonstrates the existence of a macrosomic endophenotype in autism and points toward pathogenetic links with immune dysfunctions that we speculate either lead to or are associated with increased cell cycle progression and/or decreased apoptosis.

Organophosphates (OPs) are routinely used as pesticides in agriculture and as insecticides within the household. Our prior work on Reelin and APOE delineated a gene–environment interactive model of autism pathogenesis, whereby genetically vulnerable individuals prenatally exposed to OPs during critical periods in neurodevelopment could undergo altered neuronal migration, resulting in an autistic syndrome. Since household use of OPs is far greater in the USA than in Italy, this model was predicted to hold validity in North America, but not in Europe. Here, we indirectly test this hypothesis by assessing linkage/association between autism and variants of the paraoxonase gene (PON1) encoding paraoxonase, the enzyme responsible for OP detoxification. Three functional single nucleotide polymorphisms, PON1 C−108T, L55M, and Q192R, were assessed in 177 Italian and 107 Caucasian-American complete trios with primary autistic probands. As predicted, Caucasian-American and not Italian families display a significant association between autism and PON1 variants less active in vitro on the OP diazinon (R192), according to case–control contrasts (Q192R: χ2=6.33, 1 df, P

The term "chalone" was coined about 40 years ago to describe endogenous growth-inhibiting factors with a tissue-specific and reversible effect. A large number of "chalones" were reported in the 1970's and early 80's. The term was, however, used rather indiscriminately and attempts at purification of the active component(s) were without success. As a result, most laboratories lost interest. Then, in the early 1980's, two different research teams demonstrated that the active growth-inhibiting constituents were N-substituted oligopeptides. In the following years, similar oligopeptides were characterized in extracts of liver, the large bowel, melanocytes, lymphocytes, and neuroblastoma, in addition to the two first ones from monomyelopoietic cells and epidermis, respectively. All peptides have a bell-shaped dose response pattern and optimal effect at very low concentrations. Several of them inhibit growth even in tumors derived from the respective organs. Preliminary studies have revealed that two of the oligopeptides alter the expression of growth- and differentiation-related genes. Quite recently, some papers are again using the term "chalone" and a short review therefore seems appropriate.

The colon mitosis inhibiting peptide pyroglutamyl-histidyl-glycine (pEHG) increases the expression of c-fos, fosB and egr-1 genes in the colon carcinoma cell line HT-29. However, the effect on non-tumorigenic colonic cells has not been investigated.After exposure of the cell lines YAMC (from colon mucosa of Immorto mice) and IMCE (fromn Immorto-Min mouse hybrid) to pEHG, DNA-synthesis was analysed by H3-thymidine incorporation, apoptosis and necrosis by fluorescence microscopy, and cell cycle distribution by flow cytometry.pEHG inhibited DNA-synthesis with a maximal effect at 10(-8)-10(-9) M, but stimulated at 10(-4) M. It blocked cell flow through the cell cycle at GC/M after 8 h of treatment, but had no effect on apoptosis or necrosis at any concentration. A low concentration of ascorbic acid stabilised the cells, maybe as a free radical scavanger.pEHG inhibits flux at the G2/M transition, but has no effect on cell death.

The HOXA1 gene plays a major role in brainstem and cranial morphogenesis. The G allele of the HOXA1 A218G polymorphism has been previously found associated with autism.We performed case-control and family-based association analyses, contrasting 127 autistic patients with 174 ethnically matched controls, and assessing for allelic transmission disequilibrium in 189 complete trios.A, and not G, alleles were associated with autism using both case-control (chi(2) = 8.96 and 5.71, 1 df, p.005 and.025 for genotypes and alleles, respectively), and family-based (transmission/disequilibrium test chi(2) = 8.80, 1 df, p.005) association analyses. The head circumference of 31 patients carrying one or two copies of the G allele displayed significantly larger median values (95.0th vs. 82.5th percentile, p.05) and dramatically reduced interindividual variability (p.0001), compared with 166 patients carrying the A/A genotype.The HOXA1 A218G polymorphism explains approximately 5% of the variance in the head circumference of autistic patients and represents to our knowledge the first known gene variant providing sizable contributions to cranial morphology. The disease specificity of this finding is currently being investigated. Nonreplications in genetic linkage/association studies could partly stem from the dyshomogeneous distribution of an endophenotype morphologically defined by cranial circumference.

We recently isolated and characterized a tripeptide, acetylGlu-Ser-GlyNH2 (Ac-ESG), which inhibits proliferation of lymphoid cells. In this paper we describe the effects of Ac-ESG on growth-related gene expression and cell cycle progression in two lymphoma cell lines, Ramos and Molt, representing B and T lymphocytes, respectively.RNA was extracted from Molt and Ramos cells with or without the tripeptide treatment. Gene expression was examined by semi-quantitative RT-PCR and Northern blot hybridization, and cell cycle progression was detected by flow cytometry.In the Molt cells, p53 gene expression was increased following treatment while c-myc was decreased after treatment shorter than 24 hours; N-ras expression was significantly reduced at picomolar concentration for 24-hour treatment; cyclinD1 and cdk4 expression did not show any change; DNA flow cytometry demonstrated that Molt cells were arrested or delayed predominantly in G2-M. Untreated Ramos cells had higher gene expression levels of c-myc, N-Ras and cdk4 than Molt cells, but lower p53 expression. These cells were not sensitive to Ac-ESG.The tripeptide Ac-ESG alters the expression of several growth-related genes in the Molt cell line and brings about an arrest or delay of cells in G2-M.

The intestinal mitosis-inhibiting peptide pyroglu-His-GlyOH (pEHG), inhibits normal intestinal epithelial cells and the human colon adenocarcinoma cell line HT-29 and increases the expression of c-fos (1). In this study, we investigated the mechanisms of the growth-inhibiting effects of pEHG.cDNA expression array was hybridized with cDNA from HT-29 cells exposed to pEHG or control. The results were confirmed with Northern blot or real-time PCR.pEHG(1 nM) provoked a significant increase in the expression of the early growth response protein 1 (egr-1) after an incubation of 20 minutes, while c-fos was confirmed up-regulated by the same treatment. We further studied the expression of fosB, c-jun and junB, in the AP-1 complex. fosB was up-regulated 20-fold, but only minor effects on jun variants were observed.pEHG stimulates the gene expression of some immediate-early transcription factors involved in cell proliferation.

Opioid peptides derived from food proteins (exorphins) have been found in urine of autistic patients. Based on the work of several groups, we try to show that exorphins and serotonin uptake stimulating factors may explain many of the signs and symptoms seen in autistic disorders. The individual symptoms ought to be explainable by the properties and behavioural effects of the found peptides. The data presented form the basis of an autism model, where we suggest that exorphins and serotonin uptake modulators are key mediators for the development of autism. This may be due to a genetically based peptidase deficiency in at least two or more peptidases and, or of peptidase regulating proteins made manifest by a dietary overload of exorphin precursors such as by increased gut uptake.

Neuroblastoma, the most frequent malignant tumour in neonates and young children, has an unusual clinical behaviour, age being the most important single factor. This could indicate that some growth-regulating mechanism in lost, or changed, during the first years of life. In search for possible growth-modifying factors, we identified a tetrapeptide, acetyl-Asp-Gln-Tyr-GlyNH2, in extracts of neuroblastoma tissue, in neuroblastoma cell cultures and in new-born pig brain tissue. The purified native peptide as well as a synthetic peptide with the same structure decreases in vitro growth of neuroblastoma cells at a restricted, low (picomolar) range. The structure of the peptide as well as its dose-response characteristics, indicates that it belongs to a group of endogenous growth-modifying oligopeptides that previously have been isolated from other organs and tissues. A possible role for the new peptide in clinical medicine is discussed.

The tripeptide pyroGlu-His-GlyOH (pEHG), isolated from intestinal extracts (1), suppresses growth of human colonic epithelial cells and human colorectal adenocarcinoma cells (HT-29) both in vitro and in vivo. The present study represents the first attempt to relate alterations in relevant gene expression to the effect on cell growth.Northern blot with RNA, extracted from HT-29 cells exposed to pEHG, was hybridised with c-fos and c-myc probes.c-fos gene expression was doubled in HT-29 cells after 20 minutes of incubation with pEHG and decreased to half the initial value after 2 hours. The increase at 20 minutes was concentration-dependent at a peptide concentration range from 10(-6)-10(-12) M. The expression of the oncogene c-myc showed only marginal alternations at the concentrations and times tested.The colon mitosis-inhibiting peptide pEHG increases gene expression of c-fos, but not that of c-myc.

Autism is a developmental disorder for which no cure currently exists. Gluten and/or casein free diet has been implemented to reduce autistic behaviour, in addition to special education, since early in the eighties. Over the last twelve years various studies on this dietary intervention have been published in addition to anecdotal, parental reports. The scientific studies include both groups of participants as well as single cases, and beneficial results are reported in all, but one study. While some studies are based on urinary peptide abnormalities, others are not. The reported results are, however, more or less identical; reduction of autistic behaviour, increased social and communicative skills, and reappearance of autistic traits after the diet has been broken.

The limited and predetermined number of cells that constitutes an organ or specialized cell population is to all appearances regulated according to a negative feedback principle involving growth inhibitors with sufficient tissue specificity. To find growth-inhibiting factors in lymphoid cells, we followed established purification procedures and assays. We found a single-peak fraction in water extracts of dog spleen homogenates that inhibited proliferation of Molt (T cell) lymphoma cells at low concentrations in vitro, with no significant effect on a B cell lymphoma cell line (Ramos). C-terminal amino acid sequencing and MS analysis showed the factor to be a tripeptide: N-acetyl-Glu-Ser-GlyNH(2). Treatment with a synthetic tripeptide with the structure N-acetyl-Glu-Ser-GlyNH(2) decreased the number of cell doublings of Molt cells. The peptide also delayed cell flux at the G(2)-M transition of the cell cycle, while incorporation of tritiated thymidine was not altered at the examined time points in this cell line. However, DNA synthesis in PPD-stimulated normal human lymphocytes was significantly inhibited and with a bell-shaped dose-response curve.

Adenosine deaminase (ADA) plays a relevant role in purine metabolism, immune responses, and peptidase activity, which may be altered in some autistic patients. Codominant ADA1 and ADA2 alleles code for ADA1 and ADA2 allozymes, the most frequent protein isoforms in the general population. Individuals carrying one copy of the ADA2 allele display 15 to 20% lower catalytic activity compared to ADA1 homozygotes. Recent preliminary data suggest that ADA2 alleles may be more frequent among autistic patients than healthy controls. The present study was undertaken to replicate these findings in a new case-control study, to test for linkage/association using a family-based design, and to characterize ADA2-carrying patients by serotonin blood levels, peptiduria, and head circumference. ADA2 alleles were significantly more frequent in 91 Caucasian autistic patients of Italian descent than in 152 unaffected controls (17.6% vs. 7.9%, P = 0.018), as well as among their fathers. Family-based tests involving these 91 singleton families, as well as 44 additional Caucasian-American trios, did not support significant linkage/association. However, the observed preferential maternal transmission of ADA2 alleles, if replicated, may point toward linkage disequilibrium between the ADA2 polymorphism and an imprinted gene variant located in its vicinity. Racial and ethnic differences in ADA allelic distributions, together with the low frequency of the ADA2 allele, may pose methodological problems to future linkage/association studies. Direct assessments of ADA catalytic activity in autistic individuals and unaffected siblings carrying ADA1/ADA1 vs ADA1/ADA2 genotypes may provide stronger evidence of ADA2 contributions to autistic disorder. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:784–790, 2000. © 2000 Wiley-Liss, Inc.

Oxytocin (OT), lysine vasopressin (L8VP), arginine vasopressin (A8VP) or arginine vasotocin (A8VT) are found in plasma from several species and stimulate various cell types by activation of the polyphosphoinositide metabolism and mobilization of intracellular calcium. We therefore studied the effects of the nonapeptides OT, A8VT, L8VP or A8VP on cytoplasmic calcium (Ca2+) and the effect of the nonapeptides on A8VP-induced Ca2+ mobilization. The preincubation of platelets with 'physiological' concentrations of A8VP (2-4 pmol l-1) did not enhance the intracellular calcium increase caused by the agonists used. However, the ADP-induced calcium increase was enhanced by prior addition of subthreshold concentrations of A8VP (less than 1 nmol l-1) to the platelet suspension (by 10%, P = 0.027, n = 12). Neither OT nor A8VT in concentrations from 10(-5) mumol l-1 to 1 mumol l-1 increased the cytoplasmic calcium concentration. We found that both OT and A8VT blocked the effect of subsequent exposure to A8VP. ADP (0.4 mumol l-1) did not block the effect of A8VP.

1. 1. Dohan has proposed that schizophrenia is a genetic disposition which interacts with an overload of dietary proteins such as casein and gluten or gliadin. 2. 2. A systematic attempt is made to see if this hypothesis is possible faced with aspects of schizophrenia that must be accounted for. 3. 3. The authors conclusion is that it is possible, but more serious work in this field is urgently needed.

Six schizophrenic patients had their urinary peptide levels measured before and after 5 weeks of treatment with neuroleptic medications. For two patients, levels were also measured after a reduction in the neuroleptic dose. Because of the heterogeneity of peptide peaks with the same bioactivity, the overall peptide levels were compared to initial levels. A neuroleptic effect on peptide levels was demonstrated. Several research groups have reported enzyme induction caused by neuroleptics in vivo.