Long COVID elevated MMP-9 and release from microglia by SARS-CoV-2 Spike protein.

Objective: Long COVID is a major health concern because many patients develop chronic neuropsychiatric symptoms, but the precise pathogenesis is unknown. Matrix metalloproteinase-9 (MMP-9) can disrupt neuronal connectivity and be elevated in patients with long COVID. Methods: In this study, MMP-9 was measured in the serum of long COVID patients and healthy controls, as well as in the supernatant fluid of cultured human microglia cell line stimulated by recombinant severe acute respiratory syndrome coronavirus 2 Spike protein, as well as lipopolysaccharide (LPS) and neurotensin (NT) used as positive controls. MMP-9 was measured by commercial enzyme-linked immunosorbent assay. Results: MMP-9 was significantly elevated in the serum of long COVID patients compared to healthy controls. Moreover, there was significant release of MMP-9 from a cultured human microglia cell line stimulated by LPS, NT, or Spike protein. We further show that pretreatment with the flavonoids luteolin and tetramethoxyluteolin (methlut) significantly inhibited the release of MMP-9 stimulated by the Spike protein. Conclusion: MMP-9 from Spike protein-stimulated microglia could contribute to the development of long COVID and may serve as a target for treatment including the use of luteolin.

Detection and Characterization of Extracellular Vesicles in Sputum Samples of COPD Patients.

BACKGROUND: Only one study has reported the presence of extracellular vesicles (EVs) in COPD patients' sputum. Thus, we aimed to isolate and characterize EVs from COPD and healthy individuals' sputum. METHODS: A total of 20 spontaneous sputum samples from COPD patients (m/f: 19/1) and induced sputum samples from healthy controls (m/f: 8/2) were used for EV isolation. The sputum supernatants were resuspended in PBS, precleared by centrifugation at 800× g for 10 min at 4 °C, and passed through a 0.22 µm filter (Millipore, Burlington, MA, USA). EVs were isolated by a standard membrane affinity spin column method (exoEasy maxi kit, Qiagen, Hilden, Germany). The EVs were then characterized by assessing their morphology and size using Transmission Electron Microscopy (TEM) and determining the CD9 and CD81 EV-markers with Western blot analysis. RESULTS: The EVs had a spherical shape and their mean diameter in the COPD patients was significantly greater than in the controls. Enrichment of the EV markers, CD9 and CD81, were detected in both the healthy and COPD individuals. Total EV-associated protein was significantly increased in the COPD patients compared to the controls. ROC analysis showed that total EV-associated protein in the sputum could be used to differentiate between the controls and COPD patients, with a sensitivity of 80% and a specificity of 70% at a cut-off point of 55.59 µg/mL (AUC = 0.8150). CONCLUSIONS: EVs were detectable in both the COPD and healthy individuals' sputum. The ratio of EVs in the 150-200 nm range was twice as high in the COPD patients than in the controls. The COPD patients' sputum contained increased total EV-associated protein as compared to controls, highlighting their value as a new source of specific exoproteins.

Luteolin Is More Potent than Cromolyn in Their Ability to Inhibit Mediator Release from Cultured Human Mast Cells.

INTRODUCTION: Mast cells are known for their involvement in allergic reactions but also in inflammatory reactions via secretion of numerous pro-inflammatory chemokines, cytokines, and enzymes. Drug development has focused on antiproliferative therapy for systemic mastocytosis and not on inhibitors of mast cell activation. The only drug available as a "mast cell blocker" is disodium cromoglycate (cromolyn), but it is poorly absorbed after oral administration, is a weak inhibitor of histamine release from human mast cells, and it develops rapid anaphylaxis. Instead, certain natural flavonoids, especially luteolin, can inhibit mast cell activation. METHODS: Here, we compared pretreatment (0-120 min) with equimolar concentration (effective dose for 50% inhibition = 100 mm for inhibition of histamine release by cromolyn) of cromolyn and luteolin on release of mediators from the cultured human LADR mast cell line stimulated either by immunoglobulin E (IgE) and anti-IgE or with IL-33. RESULTS: We show that luteolin is significantly more potent than cromolyn inhibiting release of histamine, tryptase, metalloproteinase-9, and vascular endothelial growth factor. Moreover, while luteolin also significantly inhibited release of IL-1ß, IL-6, and IL-8 (CXCL8) and TNF, cromolyn had no effect. CONCLUSION: These findings support the use of luteolin, especially in liposomal form to increase oral absorption, may be a useful alternative to cromolyn.

Ochratoxin A stimulates release of IL-1ß, IL-18 and CXCL8 from cultured human microglia.

Exposure to mycotoxins has been associated with the development of neuropsychiatric symptoms and Ochratoxin A (OTA) has emerged as one of the main mycotoxins associated with neurotoxicity. However, the mechanism via OTA exerts its neurotoxic effects is not well understood, especially the importance of activated microglia and their contribution to neuroinflammation. Here we report the effect of OTA on cultured immortalized human microglia-SV40, as compared to the effect of neurotensin (NT) and lipopolysaccharide (LPS) used as "positive" triggers. OTA (1, 10 and 100 nM for 24 hrs) stimulated microglia to release in the supernatant fluids statistically significant amounts of IL-1ß, IL-18 and CXCL8 assayed with ELISA. Preventing or inhibiting OTA-stimulated activation of microglia by luteolin could be an important way to limit mycotoxin-induced neuroinflammation and improve associated neuropsychiatric diseases.

Amyloid Beta Peptides Lead to Mast Cell Activation in a Novel 3D Hydrogel Model.

Alzheimer's disease (AD) is a prevalent neurodegenerative disease and the world's primary cause of dementia among the elderly population. The aggregation of toxic amyloid-beta (Aß) is one of the main pathological hallmarks of the AD brain. Recently, neuroinflammation has been recognized as one of the major features of AD, which involves a network of interactions between immune cells. The mast cell (MC) is an innate immune cell type known to serve as a first responder to pathological changes and crosstalk with microglia and neurons. Although an increased number of mast cells were found near the sites of Aß deposition, how mast cells are activated in AD is not clear. We developed a 3D culture system to culture MCs and investigated the activation of MCs by Aß peptides. Because collagen I is the major component of extracellular matrix (ECM) in the brain, we encapsulated human LADR MCs in gels formed by collagen I. We found that 3D-cultured MCs survived and proliferated at the same level as MCs in suspension. Additionally, they can be induced to secrete inflammatory cytokines as well as MC proteases tryptase and chymase by typical MC activators interleukin 33 (IL-33) and IgE/anti-IgE. Culturing with peptides Aß1-42, Aß1-40, and Aß25-35 caused MCs to secrete inflammatory mediators, with Aß1-42 inducing the maximum level of activation. These data indicate that MCs respond to amyloid deposition to elicit inflammatory responses and demonstrate the validity of collagen gel as a model system to investigate MCs in a 3D environment to understand neuroinflammation in AD.

Recombinant SARS-CoV-2 Spike Protein and Its Receptor Binding Domain Stimulate Release of Different Pro-Inflammatory Mediators via Activation of Distinct Receptors on Human Microglia Cells.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin converting enzyme 2 (ACE2) on target cells and results in acute symptoms involving especially the lungs known as COVID-19. However, increasing evidence indicates that SARS-CoV-2 infection produces neuroinflammation associated with neurological, neuropsychiatric, and cognitive symptoms persists well past the resolution of the infection, known as post-COVID-19 sequalae or long-COVID. The neuroimmune mechanism(s) involved in long-COVID have not been adequately characterized. In this study, we show that recombinant SARS-CoV-2 full-length S protein stimulates release of pro-inflammatory IL-1b, CXCL8, IL-6, and MMP-9 from cultured human microglia via TLR4 receptor activation. Instead, recombinant receptor-binding domain (RBD) stimulates release of TNF-α, IL-18, and S100B via ACE2 signaling. These results provide evidence that SARS-CoV-2 spike protein contributes to neuroinflammation through different mechanisms that may be involved in CNS pathologies associated with long-COVID.

Recombinant SARS-CoV-2 Spike Protein Stimulates Secretion of Chymase, Tryptase, and IL-1ß from Human Mast Cells, Augmented by IL-33.

SARS-CoV-2 infects cells via its spike (S) protein binding to its surface receptor angiotensin-converting enzyme 2 (ACE2) and results in the production of multiple proinflammatory cytokines, especially in the lungs, leading to what is known as COVID-19. However, the cell source and the mechanism of secretion of such cytokines have not been adequately characterized. In this study, we used human cultured mast cells that are plentiful in the lungs and showed that recombinant SARS-CoV-2 full-length S protein (1-10 ng/mL), but not its receptor-binding domain (RBD), stimulates the secretion of the proinflammatory cytokine interleukin-1ß (IL-1ß) as well as the proteolytic enzymes chymase and tryptase. The secretion of IL-1ß, chymase, and tryptase is augmented by the co-administration of interleukin-33 (IL-33) (30 ng/mL). This effect is mediated via toll-like receptor 4 (TLR4) for IL-1ß and via ACE2 for chymase and tryptase. These results provide evidence that the SARS-CoV-2 S protein contributes to inflammation by stimulating mast cells through different receptors and could lead to new targeted treatment approaches.

Exosome-associated mitochondrial DNA from patients with myalgic encephalomyelitis/chronic fatigue syndrome stimulates human microglia to release IL-1ß.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease that presents with fatigue, sleep disturbances, malaise, and cognitive problems. The pathogenesis of ME/CFS is presently unknown, and serum levels of potential biomarkers have been inconsistent. Here, we show that mitochondrial DNA (mtDNA) associated with serum exosomes, is increased in ME/CFS patients only after exercise. Moreover, exosomes isolated from patients with ME/CFS stimulate significant release of IL-1ß from cultured human microglia. These results provide evidence that activation of microglia by serum-derived exosomes may serve as a potential novel pathogenetic factor and target for treatment of ME/CFS.

ExoProK: A Practical Method for the Isolation of Small Extracellular Vesicles from Pleural Effusions.

Extracellular vesicles (EVs) are cell-secreted, lipid membrane-enclosed nanoparticles without functional nucleus. EV is a general term that includes various subtypes of particles named microvesicles, microparticles, ectosomes or exosomes. EVs transfer RNA, DNA and protein cargo between proximal and distant cells and tissues, thus constituting an organism-wide signal transduction network. Pathological tissues secrete EVs that differ in their cargo composition compared to their healthy counterparts. The detection of biomarkers in EVs from biological fluids may aid the diagnosis of disease and/or monitor its progression in a minimally invasive manner. Among biological fluids, pleural effusions (PEs) are integrated to clinical practice, as they accompany a wide variety of lung disorders. Due to the proximity with the pleura and the lungs, PEs are expected to be especially enriched in EVs that originate from diseased tissues. However, PEs are among the least studied biofluids regarding EV-specialized isolation methods and related biomarkers. Herein, we describe a practical EV isolation method from PEs for the screening of EV RNA biomarkers in clinical routine. It is based on a Proteinase K treatment step to digest contaminants prior to standard polyethylene-glycol precipitation. The efficiency of the method was confirmed by transmission electron microscopy, nanoparticle tracking analysis and Western blot. The reliability and sensitivity of the method towards the detection of EV-enriched RNA biomarkers from multiple PEs was also demonstrated.

IL-1B(3954) polymorphism and red complex bacteria increase IL-1ß (GCF) levels in periodontitis.

OBJECTIVE: The aim of this study was to compare IL-1ß levels in gingival crevicular fluid (GCF) from healthy and periodontitis sites of IL-1B(3954)-Single Nucleotide Polymorphism (SNP) positive and IL-1B(3954)-SNP negative periodontitis subjects in association with their bacterial profiles. BACKGROUND: Susceptibility to periodontitis has been associated with several risk factors, including allelic variants at multiple gene loci. Variations in the IL-1 gene cluster have been linked with increased risk for periodontitis. IL-1B(3954)-SNP has been previously associated with increased levels of IL-1ß in GCF or periodontal tissues in chronic periodontitis patients, as well as higher levels of specific periodontal pathogens. There is insufficient evidence to conclude if IL-1B gene polymorphisms affect the susceptibility to periodontitis by ultimately modulating the levels of IL-1ß in GCF, the subgingival microbial profile or both. MATERIALS AND METHODS: GCF, subgingival plaque, and buccal epithelial cells were collected from 32 individuals with periodontitis. GCF IL-1ß levels were measured by an enzyme-linked immunosorbent assay (ELISA). Bacterial plaque samples were analyzed for 11 periodontal pathogens using polymerase chain reaction (PCR) analysis with specific primers for the 16SrRNA gene of each bacterium. IL-1B(3954)-SNP status was determined by identifying the carriers of the polymorphic T allele. RESULTS: A significant association was shown between IL-1B(3954)-SNP and IL-1ß GCF levels (amount and concentration). The concomitant presence of two or three red complex bacterial species was associated with increased IL-1ß GCF levels in periodontitis sites (site-level analysis). The concurrent presence of all three red complex periodontal pathogens and IL-1B(3954)-SNP was associated with the highest IL-1ß GCF levels in periodontitis sites. CONCLUSIONS: Our results indicate an independent association of both IL-1B(3954)-SNP and red complex bacterial species with increased IL-1ß levels in GCF of periodontitis sites. A better understanding of the interaction between genetics, bacteria, and inflammation is essential to develop more effective diagnostic, prognostic, and therapeutic tools for periodontitis.

Pleural effusion osmolality correlation with pH and glucose level of pleural fluid and its effects on the pleural membrane permeability.

BACKGROUND AND AIM: Pleural effusions (PE) are a common clinical entity resulting from pathologies that affect the pleural space such as congestive heart failure, malignancy and pneumonia. The osmolality of the pleural fluid has never been studied as well as the effects of its changes on the pleural membrane. The purpose of this study was to identify the osmolality levels of PEs of different etiologies and to assess the potential effects of osmolality imbalance on the pleural permeability. MATERIALS AND METHODS: We measured the osmolality of the PEs of 64 consecutive patients (6 with transudative, 11 with parapneumonic and 47 with malignant pleural effusions) that were hospitalized in the University Hospital of Larissa. Subsequently, we selected clinically relevant hyper- and hypo- osmolality levels and performed assessment of the permeability of sheep parietal pleura by means of Ussing chamber experiments. RESULTS: The mean pleural fluid osmolality was 291.7 ± 24.89 mOms/Kg (95 % CI: 285.4-297.9), and it varied among the three groups of PEs (p = 0.05). Transformed osmolality values were associated with pH and glucose levels in the PEs. After exposure of the sheep parietal pleura to 240 mOsm/kg (hyposmolar) the transmesothelial resistance (RTM) significantly increased (p < 0.05) while at 340 mOsm/kg (hyperosmolar) the RTM was not significantly altered. CONCLUSIONS: PEs osmolality differs depending on the underlying pathology and is linked to PE pH and glucose. Hypo-osmotic PEs can lead to decreased pleural permeability. These results warrant further study of the PEs osmolality levels on the function of the pleural mesothelial cells.

IL-38 inhibits microglial inflammatory mediators and is decreased in amygdala of children with autism spectrum disorder.

Autism spectrum disorder (ASD) is characterized by impaired social interactions and communication. The pathogenesis of ASD is not known, but it involves activation of microglia. We had shown that the peptide neurotensin (NT) is increased in the serum of children with ASD and stimulates cultured adult human microglia to secrete the proinflammatory molecules IL-1ß and CXCL8. This process is inhibited by the cytokine IL-37. Another cytokine, IL-38, has been reported to have antiinflammatory actions. In this report, we show that pretreatment of cultured adult human microglia with recombinant IL-38 (aa3-152, 1-100 ng/mL) inhibits (P < 0.0001) NT-stimulated (10 nM) secretion of IL-1ß (at 1 ng/mL) and CXCL8 (at 100 ng/mL). In fact, IL-38 (aa3-152, 1 ng/mL) is more potent than IL-37 (100 ng/mL). Here, we report that pretreatment with IL-38 (100 ng/mL) of embryonic microglia (HMC3), in which secretion of IL-1ß was undetectable, inhibits secretion of CXCL8 (P = 0.004). Gene expression of IL-38 and its receptor IL-36R are decreased (P = 0.001 and P = 0.04, respectively) in amygdala from patients with ASD (n = 8) compared to non-ASD controls (n = 8), obtained from the University of Maryland NeuroBioBank. IL-38 is increased (P = 0.03) in the serum of children with ASD. These findings indicate an important role for IL-38 in the inhibition of activation of human microglia, thus supporting its development as a treatment approach for ASD.

Amyotrophic Lateral Sclerosis, Neuroinflammation, and Cromolyn.

Amyotrophic lateral sclerosis (ALS) is an upper motor neuron disease with an unknown pathogenesis and no effective treatment. A recent study found that treatment of a mouse model of ALS (TgSOD1 mice) intraperitoneally with the mast-cell blocker disodium chromoglycate (cromolyn) had a small but significant effect on disease onset, improvement of neurologic symptoms, and decrease in the expression of proinflammatory cytokines and chemokines in the spinal cord and plasma of the TgSOD1 mice. Treatment with cromolyn also reduced degranulation of mast cells in the tibialis anterior muscle. There was no effect on survival. These findings are important in their support of the involvement of mast cells in the pathogenesis of ALS but are limited by the small effect of cromolyn, which was given intraperitoneally and is poorly absorbed after oral administration. Instead, use of the structurally related flavonoid tetramethoxyluteolin, which is a more potent inhibitor of proinflammatory cytokine release from mast cells and also inhibits activated microglia, may offer significant advantages over cromolyn. Development of mast cell inhibitors could benefit not only allergic disorders but also inflammatory and neurodegenerative disorders.

Increased Expression of miR-155p5 in Amygdala of Children With Autism Spectrum Disorder.

MiR-155p5 is a pro-inflammatory microRNA reported to be involved in several neurol-inflammatory diseases. Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by impaired social interactions and communication, as well as stereotypic movements. Inflammation of the brain due to activation of microglia has been reported in ASD. We investigated miR-155p5 gene expression in postmortem human brain tissues [amygdala and dorsolateral prefrontal cortex regions (DLPFC)]. There was significant increase of miR-155p5 in amygdala (P ≤ 0.0001), but not in DLFC, in ASD children (n = 8) compared to non-ASD (n = 7) controls. The increased gene expression of miR-155p5 in amygdala of children of ASD support the presence of localized inflammation in the brain and indicates miR-155p5 may be targeted for therapy of ASD. Autism Res 2020, 13: 18-23. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: The pathogenesis of autism spectrum disorder (ASD) is still unknown. Our data of increased gene expression of miR-155p5 in brains of children with ASD support the presence of localized inflammation in the amygdala and indicates that miR-155p5 may be targeted for therapy of ASD and other neurodegenerative diseases.

IL-37 is increased in brains of children with autism spectrum disorder and inhibits human microglia stimulated by neurotensin.

Autism spectrum disorder (ASD) does not have a distinct pathogenesis or effective treatment. Increasing evidence supports the presence of immune dysfunction and inflammation in the brains of children with ASD. In this report, we present data that gene expression of the antiinflammatory cytokine IL-37, as well as of the proinflammatory cytokines IL-18 and TNF, is increased in the amygdala and dorsolateral prefrontal cortex of children with ASD as compared to non-ASD controls. Gene expression of IL-18R, which is a receptor for both IL-18 and IL-37, is also increased in the same brain areas of children with ASD. Interestingly, gene expression of the NTR3/sortilin receptor is reduced in the amygdala and dorsolateral prefrontal cortex. Pretreatment of cultured human microglia from normal adult brains with human recombinant IL-37 (1 to 100 ng/mL) inhibits neurotensin (NT)-stimulated secretion and gene expression of IL-1ß and CXCL8. Another key finding is that NT, as well as the proinflammatory cytokines IL-1ß and TNF increase IL-37 gene expression in cultured human microglia. The data presented here highlight the connection between inflammation and ASD, supporting the development of IL-37 as a potential therapeutic agent of ASD.

Mast Cells May Regulate The Anti-Inflammatory Activity of IL-37.

Mast cells are unique immune cells involved in allergic reactions, but also in immunity and inflammation. Interleukin 37 (IL-37) has emerged as an important regulatory cytokine with ability to inhibit immune and inflammatory processes. IL-37 is made primarily by macrophages upon activation of toll-like receptors (TLR) leading to generation of mature IL-37 via the action of caspase 1. In this review, we advance the premise that mast cells could regulate the anti-inflammatory activity of the IL-37 via their secretion of heparin and tryptase. Extracellular IL-37 could either dimerize in the presence of heparin and lose biological activity, or be acted upon by proteases that can generate even more biologically active IL-37 forms. Molecules that could selectively inhibit the secretion of mast cell mediators may, therefore, be used together with IL-37 as novel therapeutic agents.

Mast Cells, Neuroinflammation and Pain in Fibromyalgia Syndrome.

Fibromyalgia Syndrome (FMS) is a disorder of chronic, generalized muscular pain, accompanied by sleep disturbances, fatigue and cognitive dysfunction. There is no definitive pathogenesis except for altered central pain pathways. We previously reported increased serum levels of the neuropeptides substance P (SP) and its structural analogue hemokinin-1 (HK-1) together with the pro-inflammatory cytokines IL-6 and TNF in FMS patients as compared to sedentary controls. We hypothesize that thalamic mast cells contribute to inflammation and pain, by releasing neuro-sensitizing molecules that include histamine, IL-1ß, IL-6 and TNF, as well as calcitonin-gene related peptide (CGRP), HK-1 and SP. These molecules could either stimulate thalamic nociceptive neurons directly, or via stimulation of microglia in the diencephalon. As a result, inhibiting mast cell stimulation could be used as a novel approach for reducing pain and the symptoms of FMS.

Effects of an Extract of Salmon Milt on Symptoms and Serum TNF and Substance P in Patients With Fibromyalgia Syndrome.

PURPOSE: The aim of this study was to evaluate the effects of a dietary supplement containing primarily an extract of salmon's milt (semen) on symptoms and blood levels of proinflammatory molecules in patients with fibromyalgia syndrome (FMS), a chronic, painful musculoskeletal disease without a distinct pathogenesis or treatment. We recently reported increased serum levels of the proinflammatory molecules substance P (SP) and tumor necrosis factor (TNF) in patients with FMS as compared to those in normal controls. METHODS: This prospective, open-label study was conducted in patients with FMS (n = 87; 80 women, 7 men; age range, 18-80 years) selected from 2 clinical centers in Spain. Patients were administered the supplement and were evaluated at weeks 1 (before treatment), 4, 8, and 12 (end of treatment) for clinical parameters of functioning, fatigue, and pain, as well as overall impression. Patients were directed to take 1 capsule per day in the morning for the first 4 weeks, followed by 1 capsule in the morning and 1 capsule in the evening for the remaining 8 weeks. Differences in symptom scores in patients with FMS between weeks 1 and weeks 4, 8, and 12 were evaluated using ANOVA. Blood was obtained and serum separated in patients with FMS at 1 and 12 weeks and in a separate population of healthy controls (n = 20; 15 women, 5 men; age range, 25-65 years). Serum levels of SP and TNF were measured in patients with FMS at 1 and 12 weeks and in healthy controls by ELISA. TNF and SP levels in patients with FMS were compared between weeks 1 and 12, as well as between patients with FMS and untreated controls, using the Mann-Whitney U test. FINDINGS: Clinical parameters of functioning, fatigue, and pain, as well as overall impression, were improved significantly at 4 weeks as compared to 1 week and remained unchanged for the duration of the study (all, P < 0.0001). Serum TNF and SP levels were significantly elevated at 1 week in patients with FMS compared to controls and were decreased significantly at 12 weeks as compared to 1 week (all, P < 0.0001). IMPLICATIONS: Our findings indicate that this dietary supplement may significantly improve symptoms in patients with FMS. This is the first time to our knowledge that any molecule has been reported to be associated with a reduction in serum SP level. Consequently, the supplement or its hypothesized main active ingredient, spermine, may be developed as a novel treatment approach to FMS or other neuroinflammatory conditions. ClinicalTrials.gov identifier: NCT03911882.

Mast Cells, Stress, Fear and Autism Spectrum Disorder.

Autism Spectrum Disorder (ASD) is a developmental condition characterized by impaired communication and obsessive behavior that affects 1 in 59 children. ASD is expected to affect 1 in about 40 children by 2020, but there is still no distinct pathogenesis or effective treatments. Prenatal stress has been associated with higher risk of developing ASD in the offspring. Moreover, children with ASD cannot handle anxiety and respond disproportionately even to otherwise benign triggers. Stress and environmental stimuli trigger the unique immune cells, mast cells, which could then trigger microglia leading to abnormal synaptic pruning and dysfunctional neuronal connectivity. This process could alter the "fear threshold" in the amygdala and lead to an exaggerated "fight-or-flight" reaction. The combination of corticotropin-releasing hormone (CRH), secreted under stress, together with environmental stimuli could be major contributors to the pathogenesis of ASD. Recognizing these associations and preventing stimulation of mast cells and/or microglia could greatly benefit ASD patients.