Long COVID Syndrome Presenting as Neuropsychiatric Exacerbations in Autism Spectrum Disorder: Insights for Treatment.

COVID-19 causes not only severe respiratory symptoms, but also long-term sequelae, even if the acute-phase symptoms are minor. Neurological and neuropsychiatric symptoms are emerging as major long-term sequalae. In patients with pre-existing behavioral symptoms, such as individuals with autism spectrum disorders (ASD), the emergence of neuropsychiatric symptoms due to long COVID can be difficult to diagnose and manage. Herein, we present three ASD cases who presented with markedly worsening neuropsychiatric symptoms following COVID-19 exposure and subsequent difficulty in managing the post-COVID neuropsychiatric symptoms. Case 1 contracted SARS-CoV-2 during the early stages of the pandemic and treatment targeting COVID-19-induced immune activation was delayed. Case 2 was asymptomatic in the acute stage of a confirmed COVID-19 exposure, but still developed significant neuropsychiatric symptoms. Case 3 demonstrated a difficult course, partly due to pre-existing immune dysregulation and prior use of multiple immunomodulating agents. In cases 1 and 3 for whom serial blood samples were obtained, notable changes in the production of inflammatory and counter-regulatory cytokines by peripheral blood monocytes were observed. The presented cases illustrate the profound effects of COVID-19 on neuropsychiatric symptoms in ASD subjects and the difficulty of managing long-COVID symptoms.

Whole-exome sequencing in a subject with fluctuating neuropsychiatric symptoms, immunoglobulin G1 deficiency, and subsequent development of Crohn's disease: a case report.

BACKGROUND: Mutations or polymorphisms of genes that are associated with inflammasome functions are known to predispose individuals to Crohn's disease and likely affect clinical presentations and responses to therapeutic agents in patients with Crohn's disease. The presence of additional gene mutations/polymorphisms that can modify immune responses may further affect clinical features, making diagnosis and management of Crohn's disease even more challenging. Whole-exome sequencing is expected to be instrumental in understanding atypical presentations of Crohn's disease and the selection of therapeutic measures, especially when multiple gene mutations/polymorphisms affect patients with Crohn's disease. We report the case of a non-Hispanic Caucasian female patient with Crohn's disease who was initially diagnosed with pediatric acute-onset neuropsychiatric syndrome with fluctuating anxiety symptoms at 9 years of age. This patient was initially managed with pulse oral corticosteroid treatment and then intravenous immunoglobulin due to her immunoglobulin G1 deficiency. At 15 years of age, she was diagnosed with Crohn's disease, following onset of acute abdomen. Treatment with oral corticosteroid and then tumor necrosis factor-α blockers (adalimumab and infliximab) led to remission of Crohn's disease. However, she continued to suffer from chronic abdominal pain, persistent headache, general fatigue, and joint ache involving multiple joints. Extensive gastrointestinal workup was unrevealing, but whole-exome sequencing identified two autosomal dominant gene variants: NLRP12 (loss of function) and IRF2BP2 (gain of function). Based on whole-exome sequencing findings, infliximab was discontinued and anakinra, an interleukin-1ß blocker, was started, rendering marked improvement of her clinical symptoms. However, Crohn's disease lesions recurred following Yersinia enterocolitis. The patient was successfully treated with a blocker of interleukin-12p40 (ustekinumab), and anakinra was discontinued following remission of her Crohn's disease lesions. CONCLUSION: Loss-of-function mutation of NRLRP12 gene augments production of interleukin-1ß and tumor necrosis factor-α, while gain-of-function mutation of IRF2BP2 impairs cytokine production and B cell differentiation. We propose that the presence of these two autosomal dominant variants caused an atypical clinical presentation of Crohn's disease.

Resolution of EEG findings and clinical improvement in a patient with encephalopathy and ESES with a combination of immunomodulating agents other than corticosteroids: A case report.

Encephalopathy with electrical status epilepticus in sleep (ESES) syndrome is characterized by a near-continuous spike-and-wave discharges during sleep with marked developmental regression, mainly in speech, and the presence of clinical seizures. Although the etiology ofESES is generally unknown, its resistance to antiseizure medication (ASM), and favorable responses to oral corticosteroids (OCS), support a role for inflammation. However, the prolonged use of OCS results in undesirable side effects and alternative treatment measures are needed. Herein, we present a patient with ESES who revealed responsed to a combination of immunomodulating agents other than OCS. The patient revealed 30, 50, and 100%, reduction in the ESES pattern on EEG with the sequential addition of anakinra (interleukin-1ß inhibitor), intravenous immunoglobulin (IVIg), and sirolimus, an inhibitor of mammalian target of rapamycin (mTOR) respectively, after discontinuation of OCS due to side effects. This combination of immune-modulating agents, that were selected based on monocyte cytokine profiles, also resulted in a gradual improvement of speech and behavioral symptoms. This case indicates a possible use of immunomodulating agents other than OCS for ESES syndrome.

Serum microRNAs in ASD: Association With Monocyte Cytokine Profiles and Mitochondrial Respiration.

Our previous research has shown that purified peripheral blood monocytes (PRMo) from individuals who are diagnosed with autism spectrum disorders (ASDs) and have innate immune abnormalities reveal altered interleukin-1ß (IL-1ß)/IL-10 ratios. We also found, in separate studies, that microRNA (miRNA) expression in PBMo and mitochondrial respiration in peripheral blood mononuclear cells (PBMCs) differed in the IL-1ß/IL-10-based ASD subgroups. This study explored whether serum miRNAs are associated with both altered innate immune responses and changes in mitochondrial respiration as a link of regulatory mechanisms for these two common abnormalities in ASD subjects. Serum miRNA levels were examined by high-throughput deep sequencing in ASD and non-ASD control sera with concurrent measurement of PBMo cytokine production and mitochondrial respiration by PBMCs. ASD samples were examined as a whole group and with respect to the previously defined IL-1ß/IL-10-based ASD subgroups (high, normal, and low groups). Serum miRNA levels differed between the overall ASD sera (N = 116) and non-ASD control sera (N = 35) and also differed across the IL-1ß/IL-10-based ASD subgroups. Specifically, miRNA levels were increased and decreased in eight and nine miRNAs, respectively, in the high-ratio ASD subgroup (N = 48). In contrast, the low- (N = 25) and normal- (N = 43) ratio ASD subgroups only showed decreased miRNAs levels (18 and 10 miRNAs, respectively). Gene targets of the altered miRNAs in the high and/or low IL-1ß/IL-10 ratio ASD subgroups were enriched in pathways critical for monocyte functions and metabolic regulation. Gene targets of the altered miRNAs in all the ASD subgroups were enriched in pathways of neuronal development and synaptic plasticity, along with cell proliferation/differentiation. ASD subgroup-specific associations were observed between serum miRNA expression and IL-1ß/IL-10 ratios, mitochondrial respiration, and monocyte cytokine profiles (IL-10, CCL2, and TNF-α). In summary, our results indicate that serum levels of select miRNAs may serve as promising biomarkers for screening and monitoring changes in innate immunity and mitochondrial respiration in ASD.

Associations between Monocyte and T Cell Cytokine Profiles in Autism Spectrum Disorders: Effects of Dysregulated Innate Immune Responses on Adaptive Responses to Recall Antigens in a Subset of ASD Children.

Changes in monocyte cytokine production with toll like receptor (TLR) agonists in subjects with autism spectrum disorders (ASD) were best reflected by the IL-1ß/IL-10 ratios in our previous research. The IL-1ß/IL-10 based subgrouping (low, normal, and high) of ASD samples revealed marked differences in microRNA expression, and mitochondrial respiration. However, it is unknown whether the IL-1ß/IL-10 ratio based subgrouping is associated with changes in T cell cytokine profiles or monocyte cytokine profiles with non-TLR agonists. In ASD (n = 152) and non-ASD (n = 41) subjects, cytokine production by peripheral blood monocytes (PBMo) with TLR agonists and ß-glucan, an inflammasome agonist, and T cell cytokine production by peripheral blood mononuclear cells (PBMCs) with recall antigens (Ags) (food and candida Ags) were concurrently measured. Changes in monocyte cytokine profiles were observed with ß-glucan in the IL-1ß/IL-10 ratio based ASD subgroups, along with changes in T cell cytokine production and ASD subgroup-specific correlations between T cell and monocyte cytokine production. Non-ASD controls revealed considerably less of such correlations. Altered innate immune responses in a subset of ASD children are not restricted to TLR pathways and correlated with changes in T cell cytokine production. Altered trained immunity may play a role in the above described changes.

Variations in Mitochondrial Respiration Differ in IL-1ß/IL-10 Ratio Based Subgroups in Autism Spectrum Disorders.

Autism spectrum disorder (ASD)7 is associated with multiple physiological abnormalities, including immune dysregulation, and mitochondrial dysfunction. However, an association between these two commonly reported abnormalities in ASD has not been studied in depth. This study assessed the association between previously identified alterations in cytokine profiles by ASD peripheral blood monocytes (PBMo) and mitochondrial dysfunction. In 112 ASD and 38 non-ASD subjects, cytokine production was assessed by culturing purified PBMo overnight with stimuli of innate immunity. Parameters of mitochondrial respiration including proton-leak respiration (PLR), ATP-linked respiration (ALR), maximal respiratory capacity (MRC), and reserve capacity (RC) were measured in peripheral blood mononuclear cells (PBMCs). The ASD samples were analyzed by subgrouping them into high, normal, and low IL-1ß/IL-10 ratio groups, which was previously shown to be associated with changes in behaviors and PBMo miRNA expression. MRC, RC, and RC/PLR, a marker of electron transport chain (ETC) efficiency, were higher in ASD PBMCs than controls. The expected positive associations between PLR and ALR were found in control non-ASD PBMCs, but not in ASD PBMCs. Higher MRC, RC, RC/PLR in ASD PBMCs were secondary to higher levels of these parameters in the high and normal IL-1ß/IL-10 ratio ASD subgroups than controls. Associations between mitochondrial parameters and monocyte cytokine profiles differed markedly across the IL-1ß/IL-10 ratio based ASD subgroups, rendering such associations less evident when ASD samples as a whole were compared to non-ASD controls. Our results indicate for the first time, an association between PBMC mitochondrial function and PBMo cytokine profiles in ASD subjects. This relationship differs across the IL-1ß/IL-10 ratio based ASD subgroups. Changes in mitochondrial function are likely due to adaptive changes or mitochondrial dysfunction, resulting from chronic oxidative stress. These results may indicate alteration in molecular pathways affecting both the immune system and mitochondrial function in some ASD subjects.

MicroRNA expression changes in association with changes in interleukin-1ß/interleukin10 ratios produced by monocytes in autism spectrum disorders: their association with neuropsychiatric symptoms and comorbid conditions (observational study).

BACKGROUND: MicroRNAs (miRNAs) play a major role in regulating immune responses at post-transcriptional levels. Previously, we have reported fluctuating interlukine-1ß (IL-1ß)/IL-10 ratios produced by peripheral blood monocytes (PBMo) in some patients with autism spectrum disorders (ASD). This study examined whether changes in miRNA expression by PBMo are associated with changes in IL-1ß/IL-10 ratios and how such changes are associated with ASD clinical features. METHODS: miRNA expression by purified PBMo from ASD subjects (N = 69) and non-ASD controls (N = 27) were determined by high-throughput sequencing. Cytokine production by PBMo in responses to stimuli of innate immunity, and behavioral symptoms [assessed by aberrant behavioral checklist (ABC)] were also evaluated at the same time of sample obtainment. RESULTS: As a whole, there was no difference in miRNA expression between ASD and control non-ASD PBMo. However, when ASD cells were subdivided into 3 groups with high, normal, or low IL-1ß/IL-10 ratios as defined in the "Results" section, in comparison with the data obtained from non-ASD controls, we observed marked changes in miRNA expression. Namely, over 3-fold changes in expression of miR-181a, miR-93, miR-223, miR-342, and miR-1248 were observed in ASD PBMo with high or low IL-1ß/IL-10 ratios, but not in ASD PBMo with normal ratios. These miRNAs that had altered in expression are those closely associated with the regulation of key signaling pathways. With changes in IL-1ß/IL-10 ratios, we also observed changes in the production of cytokines (IL-6, TNF-α, and TGF-ß) other than IL-1ß/IL-10 by ASD PBMo. The association between behavioral symptoms and cytokine levels was different when ASD cells exhibit high/low IL-1ß/IL-10 ratios vs. when ASD cells exhibited normal ratios. Non-IgE-mediated food allergy was also observed at higher frequency in ASD subjects with high/low IL-1ß/IL-10 ratios than with normal ratios. CONCLUSIONS: Changes in cytokine profiles and miRNA expression by PBMo appear to be associated with changes in ASD behavioral symptoms. miRNAs that are altered in expression in ASD PBMo with high/low IL-1ß/IL-10 ratios are those associated with inflammatory responses. Changes in IL-1ß/IL-10 ratios along with changes in miRNA expression may serve as biomarkers for immune-mediated inflammation in ASD.

Practically feasible production of sustained-release microspheres of granulocyte-macrophage colony-stimulating factor (rhGM-CSF).

Using recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) as a model drug, the present study demonstrated a practically feasible process to produce polymeric microspheres for sustained-release delivery of protein drugs with preserved integrity. This process is featured with pre-loading proteins into polysaccharide fine particles via a self-standing aqueous-aqueous "emulsion", prior to microencapsulation into the microspheres. The protein drug, rhGM-CSF, was partitioned thermodynamically into a dextran dispersed phase of the aqueous-aqueous emulsion, followed by lyophilization and removal of the polyethylene glycol (PEG) continuous phase (using an organic solvent not penetrating into dextran matrix). The harvested dextran particles were then suspended in a dichloromethane solution of polylatic-co-glyclic acids (PLGA) and emulsified in a polyvinyl alcohol (PVA) and NaCl solution of small volume to form embryonic microspheres. The emulsion was then transferred into a NaCl solution of large volume to extract the organic solvent and harden the embryonic microspheres. The obtained rhGM-CSF microspheres showed a satisfied release profile with the day-to-day variation within 9 folds over the multi-weeks long release period. At the same time, the integrity (defined freedom of aggregates measured by SEC-HPLC) and bioactivity (defined by TF-1 cell proliferation) of the proteins were well preserved. The present formulation process ensured good reproducibility and over 89% protein encapsulation efficiency, and practically feasible to adapt to scaled productions.

Cytokine profiles by peripheral blood monocytes are associated with changes in behavioral symptoms following immune insults in a subset of ASD subjects: an inflammatory subtype?

BACKGROUND: Some children with autism spectrum disorders (ASD) are characterized by fluctuating behavioral symptoms following immune insults, persistent gastrointestinal (GI) symptoms, and a lack of response to the first-line intervention measures. These children have been categorized as the ASD-inflammatory subtype (ASD-IS) for this study. We reported a high prevalence of non-IgE mediated food allergy (NFA) in young ASD children before, but not all ASD/NFA children reveal such clinical features of ASD-IS. This study addressed whether behavioral changes of ASD-IS are associated with innate immune abnormalities manifested in isolated peripheral blood (PB) monocytes (Mo), major innate immune cells in the PB. METHODS: This study includes three groups of ASD subjects (ASD-IS subjects (N = 24), ASD controls with a history of NFA (ASD/NFA (N = 20), and ASD/non-NFA controls (N = 20)) and three groups of non-ASD controls (non-ASD/NFA subjects (N = 16), those diagnosed with pediatric acute onset-neuropsychiatric syndrome (PANS, N = 18), and normal controls without NFA or PANS (N = 16)). Functions of purified PB Mo were assessed by measuring the production of inflammatory and counter-regulatory cytokines with or without stimuli of innate immunity (lipopolysaccharide (LPS), zymosan, CL097, and candida heat extracts as a source of ß-lactam). In ASD-IS and PANS subjects, these assays were done in the state of behavioral exacerbation ('flare') and in the stable ('non-flare') condition. ASD-IS children in the 'flare' state revealed worsening irritability, lethargy and hyperactivity. RESULTS: 'Flare' ASD-IS PB Mo produced higher amounts of inflammatory cytokines (IL-1ß and IL-6) without stimuli than 'non-flare' ASD-IS cells. With zymosan, 'flare' ASD-IS cells produced more IL-1ß than most control cells, despite spontaneous production of large amounts of IL-1ß. Moreover, 'flare' ASD-IS Mo produced less IL-10, a counterregulatory cytokine, in response to stimuli than 'non-flare' cells or other control cells. These changes were not observed in PANS cells. CONCLUSIONS: We observed an imbalance in the production of inflammatory (IL-1ß and IL-6) and counterregulatory (IL-10) cytokines by 'flare' ASD-IS monocytes, which may indicate an association between intrinsic abnormalities of PB Mo and changes in behavioral symptoms in the ASD-IS subjects.

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.

INTRODUCTION: There 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 DESCRIPTION: We 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 EVALUATION: We 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. CONCLUSIONS: The 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.

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.

Age-dependent changes in peripheral blood dendritic cell subsets in normal children and children with specific polysaccharide antibody deficiency (SPAD).

Myeloid and plasmacytoid dendritic cells (MDC/PDC) play crucial roles in bridging adaptive and innate immunity by affecting development of both cellular and humoral immunity. The immune system evolves after birth as reflected in dynamic changes in numbers and functions of various immune cells with age. However, age-associated changes in DC subsets in children have not been elucidated despite the fact that such normative data are crucial for evaluating alternations of DC subsets in various pediatric diseases. This study addressed age-associated changes in DC subsets and CD40/86 expression on PDC (markers of maturation/activation) in 50 healthy children in comparison with 25 children with specific polysaccharide antibody deficiency (SPAD). Our results revealed age-dependent decrease of PDC numbers (p < 0.0001), although there was no age-associated changes in CD40/CD86 expression. MDC1/MDC2 numbers did not reveal such linear age-dependent changes and MDC1/PDC ratio reached around 2 as typically seen in young adults after 10 years of age. In contrast, SPAD patients did not reveal such age-associated changes and showed decreased fluorescence intensity of CD86 in PDC cells. These results indicate lineage specific, age-dependent changes in DC subsets in normal children and possible altered development of these cells in SPAD children, emphasizing the importance of age-appropriate controls.

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.

Toll like receptors (TLR) regulate innate immune responses sensing byproducts of intestinal microbiota. We examined responses to TLR agonists in children with inflammatory bowel disease (IBD). Peripheral blood mononuclear cells (PBMC) obtained from children with IBD [Crohn's disease (CD, n = 10), ulcerative colitis (UC, n = 10)], children with non-IgE-mediated food allergy (NFA, n = 20), and controls (n = 15) were tested for their production of proinflammatory and counter-regulatory cytokines with TLR agonists in comparison with their cytokine production against milk protein and candida. IBD patients were all in the inactive state. IBD PBMC produced more IL-6 with all the TLR agonists tested than controls. CD PBMC produced more counter-regulatory cytokines with TLR agonists, while UC PBMC produced more IL-1ss and IL-10 with TLR 7/8 agonist than controls. Cytokine production by NFA PBMC did not differ from controls. CD but not UC PBMC produced more IFN-gamma and IL-17 with candida. Aberrant responses to TLR agonists may be associated with increase in IFN-gamma/IL-17 production against candida in CD children.

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

BACKGROUND: Among 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. METHODS: This 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. RESULTS: Non-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-1beta 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-1beta with TLR4/7/8 agonists with LPS pre-treatment. These are cytokines associated with the neuro-immune network. CONCLUSION: Clinical 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.

Preparing polymer-based sustained-release systems without exposing proteins to water-oil or water-air interfaces and cross-linking reagents.

We report a method to load proteins into polymer-based sustained-release systems without exposing them to water-oil or water-air interfaces, factors known to denature proteins. By dispersing a dextan solution containing a protein into a PEG solution containing small amount of alginate, a stable aqueous-aqueous "emulsion" was formed. The poly-anionic alginate generated a diffuse double layer around each dextran droplet to prevent them from contacting with each other and fusing to a block phase. Proteins distributed in the stabilized dextran droplets by preferential partition favoring dextran. Freeze-drying this emulsion resulted in protein-loaded dextran particles, 1-2 microm in diameter and 1.6 g/cm(3) in density. The particles were harvested by washing the lyophilized powder using organic solvents to remove the PEG continuous phase. An activity assay of encapsulated beta-galactosidase indicated that protein activity was preserved during the particle-forming process including the step of sonicating the particles in organic solvents. The dextran particles also improved release profile and integrity of proteins when encapsulated in degradable polymer sustained-release systems. The aqueous-aqueous emulsion offers a convenient way to prepare solvent-resistant protein-polysaccharide particles that can easily be incorporated in a variety of polymer-based pharmaceutical dosage forms and medical devices such as microspheres, scaffolds and drug-eluting stents for sustained-release protein delivery.

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

UNLABELLED: 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.

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

We present a case of fetal sensitization to cow's 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-alpha (TNF-alpha) production by cord blood mononuclear cells. Following dietary intervention, we observed a decline of TNF-alpha 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.

Evaluation of an association between gastrointestinal symptoms and cytokine production against common dietary proteins in children with autism spectrum disorders.

OBJECTIVE: To evaluate an association between cytokine production with common dietary proteins as a marker of non-allergic food hypersensitivity (NFH) and gastrointestinal (GI) symptoms in young children with autism spectrum disorders (ASD). STUDY DESIGN: Peripheral blood mononuclear cells (PBMCs) were obtained from 109 ASD children with or without GI symptoms (GI [+] ASD, N = 75 and GI (-) ASD, N = 34], from children with NFH (N = 15), and control subjects (N = 19). Diarrhea and constipation were the major GI symptoms. We measured production of type 1 T-helper cells (Th1), type 2 T-helper cells (Th2), and regulatory cytokines by PBMCs stimulated with whole cow's milk protein (CMP), its major components (casein, beta-lactoglobulin, and alpha-lactoalbumin), gliadin, and soy. RESULTS: PBMCs obtained from GI (+) ASD children produced more tumor necrosis factor-alpha (TNF-alpha)/interleukin-12 (IL-12) than those obtained from control subjects with CMP, beta-lactoglobulin, and alpha-lactoalbumin, irrespective of objective GI symptoms. They also produced more TNF-alpha with gliadin, which was more frequently observed in the group with loose stools. PBMCs obtained from GI (-) ASD children produced more TNF-alpha/IL-12 with CMP than those from control subjects, but not with beta-lactoglobulin, alpha-lactoalbumin, or gliadin. Cytokine production with casein and soy were unremarkable. CONCLUSION: A high prevalence of elevated TNF-alpha/IL-12 production by GI (+) ASD PBMCs with CMP and its major components indicates a role of NFH in GI symptoms observed in children with ASD.

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-alpha, IL-1beta, 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-alpha 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-alpha 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.

Triple immune suppression increases short-term survival of porcine fetal retinal pigment epithelium xenografts.

PURPOSE: To determine the effect of triple drug immune suppression on RPE xenograft survival in the fetal pig after transplantation into the albino rabbit subretinal space. METHODS: Primary RPE microaggregates (approximately 40,000 RPE cells) were injected into the subretinal space of 24 albino rabbits, with half the rabbits maintained on triple systemic immune suppression. RPE survival was estimated with a DNA probe (porcine DNA repeat element; PRE) against a porcine-specific repetitive chromosomal marker or a RAM-11 antibody against rabbit macrophages. RESULTS: Numerous pigmented cells were visible in the subretinal space at all time points, but most pigment-containing cells 4 weeks or more after surgery were RAM-11 positive and PRE negative. The number of PRE-positive cells in the immune-suppressed group (4193 +/- 2461, 1184 +/- 1502, and 541 +/- 324 at 4, 8, and 12 weeks, respectively) was greater than in immune-competent control animals (292 +/- 506, 193 +/- 173, and 111 +/- 96), but the difference was only statistically significant at 4 weeks. The time-dependent decrease in PRE-positive cells was more pronounced in immune-suppressed animals. Image analysis performed on serial fundus photographs and fluorescein angiograms did not detect any difference in the appearance of the grafts in immune-suppressed versus immune-competent animals. CONCLUSIONS: Systemic immune suppression increased the 4-week survival of porcine RPE xenografts in the albino rabbit subretinal space, but there was poor survival in immune-suppressed and -competent animals 12 weeks after surgery. Many pigment-containing cells 4 or more weeks after surgery were PRE negative, indicating that they are of host origin.