Lee Geng

Dysregulated Innate Immune Responses in Young Children with Autism Spectrum Disorders: Their Relationship to Gastrointestinal Symptoms and Dietary Intervention

Objective: Our previous study indicated an association between cellular immune reactivity to common dietary proteins (DPs) and excessive proinflammatory cytokine production with endotoxin (lipopolysaccharide, LPS), a major stimulant of innate immunity in the gut mucosa, in a subset of autism spectrum disorder (ASD) children. However, it is unclear whether such abnormal LPS responses are intrinsic in these ASD children or the results of chronic gastrointestinal (GI) inflammation secondary to immune reactivity to DPs. This study further explored possible dysregulated production of proinflammatory and counter-regulatory cytokines with LPS in ASD children and its relationship to GI symptoms and the effects of dietary intervention measures. Methods: This study includes ASD children (median age 4.8 years) on the unrestricted (n = 100) or elimination (n = 77) diet appropriate with their immune reactivity. Controls include children with non-allergic food hypersensitivity (NFH; median age 2.9 years) on the unrestricted (n = 14) or elimination (n = 16) diet, and typically developing children (median age 4.5 years, n = 13). The innate immune responses were assessed by measuring production of proinflammatory (TNF-α, IL-1β, IL-6, and IL-12) and counter-regulatory (IL-1ra, IL-10, and sTNFRII) cytokines by peripheral blood mononuclear cells (PBMCs) with LPS. The results were also compared to T-cell responses with common DPs and control T-cell mitogens assessed by measuring T-cell cytokine production. Results: ASD and NFH PBMCs produced higher levels of TNF-α with LPS than controls regardless of dietary interventions. However, only in PBMCs from ASD children with positive gastrointestinal (GI(+)) symptoms, did we find a positive association between TNF-α levels produced with LPS and those with cow’s milk protein (CMP) and its major components regardless of dietary interventions. In the unrestricted diet group, GI(+) ASD PBMCs produced higher IL-12 than controls and less IL-10 than GI(–) ASD PBMCs with LPS. GI(+) ASD but not GI(–) ASD or NFH PBMCs produced less counter-regulatory cytokines with LPS in the unrestricted diet group than in the elimination diet group. There was no significant difference among the study groups with regard to cytokine production in responses to T-cell mitogens and other recall antigens. Conclusion: Our results revealed that there are findings limited to GI(+) ASD PBMCs in both the unrestricted and elimination diet groups. Thus our findings indicate intrinsic defects of innate immune responses in GI(+) ASD children but not in NFH or GI(–) ASD children, suggesting a possible link between GI and behavioral symptoms mediated by innate immune abnormalities.

Impact of innate immunity in a subset of children with autism spectrum disorders: a case control study

BackgroundAmong patients with autism spectrum disorders (ASD) evaluated in our clinic, there appears to be a subset that can be clinically distinguished from other ASD children because of frequent infections (usually viral) accompanied by worsening behavioural symptoms and/or loss/decrease in acquired skills. This study assessed whether these clinical features of this ASD subset are associated with atopy, asthma, food allergy (FA), primary immunodeficiency (PID), or innate immune responses important in viral infections.MethodsThis study included the ASD children described above (ASD test, N = 26) and the following controls: ASD controls (N = 107), non-ASD controls with FA (N = 24), non-ASD controls with chronic rhinosinusitis/recurrent otitis media (CRS/ROM; N = 38), and normal controls (N = 43). We assessed prevalence of atopy, asthma, FA, CRS/ROM, and PID. Innate immune responses were assessed by measuring production of proinflammatory and counter-regulatory cytokines by peripheral blood mononuclear cells (PBMCs) in response to agonists of Toll-like receptors (TLRs), with or without pre-treatment of lipopolysaccharide (LPS), a TLR4 agonist.ResultsNon-IgE mediated FA was equally prevalent in both ASD test and ASD control groups, occurring at higher frequency than in the non-ASD controls. Allergic rhinitis, atopic/non-atopic asthma, and atopic dermatitis were equally prevalent among the study groups except for the CRS/ROM group in which non-atopic asthma was more prevalent (52.6%). CRS/ROM and specific polysaccharide antibody deficiency (SPAD) were more prevalent in the ASD test group than in the ASD control, FA, and normal control groups: 23.1% vs. < 5% for CRS/ROS and 19.2% vs. < 1% for SPAD. However, CRS/ROM patients had the highest prevalence of SPAD (34.2%). When compared to ASD and normal case controls, PBMCs from 19 non-SPAD, ASD test group children produced: 1) less IL-1β with a TLR7/8 agonist, less IL-10 with a TLR2/6 agonist, and more IL-23 with a TLR4 agonist without LPS pre-treatment, and 2) less IL-1β with TLR4/7/8 agonists with LPS pre-treatment. These are cytokines associated with the neuro-immune network.ConclusionClinical features of the ASD test group were not associated with atopy, asthma, FA, or PID in our study but may be associated with altered TLR responses mediating neuro-immune interactions.

Children with autism spectrum disorders (ASD) who exhibit chronic gastrointestinal (GI) symptoms and marked fluctuation of behavioral symptoms exhibit distinct innate immune abnormalities and transcriptional profiles of peripheral blood (PB) monocytes

Innate/adaptive immune responses and transcript profiles of peripheral blood monocytes were studied in ASD children who exhibit fluctuating behavioral symptoms following infection and other immune insults (ASD/Inf, N=30). The ASD/Inf children with persistent gastrointestinal symptoms (ASD/Inf+GI, N=19), revealed less production of proinflammatory and counter-regulatory cytokines with stimuli of innate immunity and marked changes in transcript profiles of monocytes as compared to ASD/no-Inf (N=28) and normal (N=26) controls. This included a 4-5 fold up-regulation of chemokines (CCL2 and CCL7), consistent with the production of more CCL2 by ASD/Inf+GI cells. These results indicate dysregulated innate immune defense in the ASD/Inf+GI children, rendering them more vulnerable to common microbial infection/dysbiosis and possibly subsequent behavioral changes.

Immunological characterization and transcription profiling of peripheral blood (PB) monocytes in children with autism spectrum disorders (ASD) and specific polysaccharide antibody deficiency (SPAD): case study

IntroductionThere exists a small subset of children with autism spectrum disorders (ASD) characterized by fluctuating behavioral symptoms and cognitive skills following immune insults. Some of these children also exhibit specific polysaccharide antibody deficiency (SPAD), resulting in frequent infection caused by encapsulated organisms, and they often require supplemental intravenous immunoglobulin (IVIG) (ASD/SPAD). This study assessed whether these ASD/SPAD children have distinct immunological findings in comparison with ASD/non-SPAD or non-ASD/SPAD children.Case descriptionWe describe 8 ASD/SPAD children with worsening behavioral symptoms/cognitive skills that are triggered by immune insults. These ASD/SPAD children exhibited delayed type food allergy (5/8), treatment-resistant seizure disorders (4/8), and chronic gastrointestinal (GI) symptoms (5/8) at high frequencies. Control subjects included ASD children without SPAD (N = 39), normal controls (N = 37), and non-ASD children with SPAD (N = 12).Discussion and EvaluationWe assessed their innate and adaptive immune responses, by measuring the production of pro-inflammatory and counter-regulatory cytokines by peripheral blood mononuclear cells (PBMCs) in responses to agonists of toll like receptors (TLR), stimuli of innate immunity, and T cell stimulants. Transcription profiling of PB monocytes was also assessed. ASD/SPAD PBMCs produced less proinflammatory cytokines with agonists of TLR7/8 (IL-6, IL-23), TLR2/6 (IL-6), TLR4 (IL-12p40), and without stimuli (IL-1ß, IL-6, and TNF-α) than normal controls. In addition, cytokine production of ASD/SPAD PBMCs in response to T cell mitogens (IFN-γ, IL-17, and IL-12p40) and candida antigen (Ag) (IL-10, IL-12p40) were less than normal controls. ASD/non-SPAD PBMDs revealed similar results as normal controls, while non-ASD/SPAD PBMCs revealed lower production of IL-6, IL-10 and IL-23 with a TLR4 agonist. Only common features observed between ASD/SPAD and non-ASD/SPAD children is lower IL-10 production in the absence of stimuli. Transcription profiling of PB monocytes revealed over a 2-fold up (830 and 1250) and down (653 and 1235) regulation of genes in ASD/SPAD children, as compared to normal (N = 26) and ASD/non-SPAD (N = 29) controls, respectively. Enriched gene expression of TGFBR (p < 0.005), Notch (p < 0.01), and EGFR1 (p < 0.02) pathways was found in the ASD/SPAD monocytes as compared to ASD/non-SPAD controls.ConclusionsThe Immunological findings in the ASD/SPAD children who exhibit fluctuating behavioral symptoms and cognitive skills cannot be solely attributed to SPAD. Instead, these findings may be more specific for ASD/SPAD children with the above-described clinical characteristics, indicating a possible role of these immune abnormalities in their neuropsychiatric symptoms.

Monozygous twins with a microdeletion syndrome involving BTK, DDP1, and two other genes; evidence of intact dendritic cell development and TLR responses.

We report for the first time monozygous twins with a microdeletion syndrome involving genes coding for Bruton’s tyrosine kinase (Btk) and deafness-dystonia peptide 1 (DDP1), and two other genes. Apart from its essential role in B cell development, Btk is indicated to affect signaling mediated by toll like receptors (TLRs) and development of dendritic cells (DCs) but results are conflictive. The twins revealed normal numbers of plasmacytoid and myeloid DCs (pDCs and mDCs). Moreover, BTK null cells from these patients exhibited robust responses to TLR agonists, normal natural killer (NK) cell activity, and normal pDC functions.Conclusion: Our results do not indicate the essential role of Btk in TLR signaling and DC development.

Aberrant responses to TLR agonists in pediatric IBD patients; the possible association with increased production of Th1/Th17 cytokines in response to candida, a luminal antigen

Jyonouchi H, Geng L, Cushing‐Ruby A, Monteiro IM. Aberrant responses to TLR agonists in pediatric IBD patients; the possible association with increased production of Th1/Th17 cytokines in response to candida, a luminal antigen.
Pediatr Allergy Immunol 2010: 21: e747–e755.
© 2009 John Wiley & Sons A/S

Intractable Epilepsy (IE) and Responses to Anakinra, a Human Recombinant IL-1 Receptor Agonist (IL-1ra): Case Reports

Patients with intractable seizures (IE) who failed to respond currently available treatment measures are clinically challenging, causing significant morbidity and even mortality. Better understanding of contributing factors for IE will be helpful for exploring additional treatment options. A role of inflammation has long been suspected in pathogenesis of epilepsy, especially in idiopathic epilepsy with diffuse epileptic activity. In rodent models of epilepsy, blockage of inflammatory mediators such as interleukin 1-β (IL-1β) and IL-6 attenuates epileptic activities. However, therapeutic effects of blockers of such inflammatory mediators have not been addressed in patients with IE.This manuscript reports 4 IE cases in which anakira, a human recombinant IL-1 receptor antagonist (IL-1ra), was clinically effective in controlling their epileptic activity in a dose-dependent manner. In these patients, we also observed changes in ratios of IL-1β/IL-10 produced by peripheral blood monocytes, surrogates of bone marrow derived microglial cells. These findings could be associated with seizure control status.These presented cases indicate a possible therapeutic use of a commercially available IL-1β blocker (anakinra) and therapeutic utility of the IL-1β/IL-10 ratios produced by peripheral blood monocytes for assessing responses to this immune-modulating agent.

Fetal sensitization to cow's milk protein and wheat: cow's milk protein and wheat-specific TNF-α production by umbilical cord blood cells and subsequent decline of TNF-α production by peripheral blood mononuclear cells following dietary intervention

We present a case of fetal sensitization to cows milk protein (CMP) and wheat, resulting in non‐IgE mediated food allergy (NFA). Fetal sensitization was indicated by onset of NFA symptoms shortly after birth and CMP/wheat‐specific tumor necrosis factor‐α (TNF‐α) production by cord blood mononuclear cells. Following dietary intervention, we observed a decline of TNF‐α production by peripheral blood mononuclear cells with stimuli of these dietary proteins (DPs) but recurrence of reactivity was observed following viral gastroenteritis, while interleukin‐10 production with these DPs persisted during his first 5 yr of life. This finding may indicate active suppressive mechanisms for maintaining oral tolerance.

Interleukin-1 β /Interleukin10 Ratio Produced by Monocytes as a Biomarker of Neuroinflammation in Autism

Objective: Innate immune abnormalities have been frequently reported in children with autism spectrum disorders (ASD), but a role of innate immunity in ASD is not well understood. This study explored a possible role of innate immunity in ASD clinical features and co-morbidities.Methods: Purified peripheral blood monocytes (PBMo) from ASD (N=125) and non-ASD (N=36) subjects were cultured overnight with or without stimulants of innate immunity, and production of pro-inflammatory and counter-regulator cytokines were assessed. Behavioral symptoms were assessed by aberrant behavioral checklist (ABC) at the time of PBMo sampling.Results: ASD PBMo revealed highly variable IL-1β/IL-10 ratios, in contrast to a tight range of IL-1β/IL-10 ratios in non-ASD control cells. There was no association between cytokine levels or IL-1β/IL-10 ratios and ABC subscale scores when ASD data was analyzed, as a whole. However, when ASD data was separated into high, low, or normal (equivalent to controls) IL-1β/IL-1 ratio groups, IL-1β levels were positively associated with stereotypy in the high ratio group. In contrast, IL-1β and IL-10 levels were negatively associated with irritability, lethargy, and hyperactivity in the normal ratio group. The low ratio group revealed a negative association between IL-1β levels and lethargy. When longitudinal changes in cytokine production from PBMo were studied in selected ASD subjects, fluctuating ratios were found in ASD subjects with deviated (high or low) IL-1β/IL-10 ratios, but ratios remained stable in ASD subjects with normal ratios. ASD subjects with deviated ratios were found to have higher frequencies of non-IgE mediated food allergy (NFA) (p<0.05) and seizure disorder (p<0.01) than those with normal ratios.Conclusion: IL-1β and IL-10 produced by innate immune responses play crucial roles in the neuroimmune network. Thus the deviated (high or low) IL-1β/IL-10 ratio from ASD monocytes may be a promising candidate biomarker for assessing changes of neuroimmune regulations and risk of comorbidities (NFA and seizure disorders) in ASD.

Impaired Oral Tolerance in ASD Children with Food Protein Induced Enterocolitis Syndrome (FPIES) – Altered Innate and Adaptive Immune Responses in ASD Children Recovered from FPIES in Comparison with non-ASD/FPIES and ASD/non-FPIES Children

The gut mucosal immune system has to maintain an intricate immune homeostasis by maintaining tolerance to macronutrients, commensal flora, and other harmless molecules in the gut lumen while exerting effective immune defense against pathogenic microbes. It takes the first few years of life to establish this intricate immune homeostasis and until then the gut mucosal immune system is rather error-prone. This is reflected in the fact that young infants and children often suffer from temporary intolerance to common food proteins (FP), e.g. food allergy (FA). During this period, most FA manifests as delayed type FA, mediated by cellular immune responses – often called FP induced enterocolitis syndrome (FPIES) (Jyonouchi, 2008; Nowak-Wegrzyn and Muraro, 2009; Sicherer et al., 1998). Although FPIES seldom cause fatal anaphylaxis, as opposed to IgE-mediated FA, this condition is often under-diagnosed and under-treated. This is, in part, due to the delayed onset of symptoms that renders clinical connection more difficult, as well as, the lack of commercially available diagnostic measures. This is in contrast to IgE mediated FA in which a causal relationship is apparent in many cases and reactivity to food allergens is easily detected with prick skin test (PST) and/or presence of FA specific IgE antibody. The delay in the onset of symptoms of FPIES makes it especially difficult to diagnose FPIES clinically in infants and young children, as well as those with limited expressive language including children with autism spectrum disorders (ASD). In general, FPIES has an excellent prognosis provided there is timely implementation of avoidance measures against offending food (Jyonouchi, 2008; Nowak-Wegrzyn and Muraro, 2009). However, delayed diagnosis/treatment of FPIES can lead to failure to thrive (FTT), protein losing enteropathy, and possibly other irreversible complications.