**1. Introduction**

Autism spectrum disorder (ASD) is a behavioral defined syndrome except for a small subset of patients with defined gene mutations such as MECP2 (Rett syndrome) and TSC1/TSC2 (tuberous sclerosis) [1, 2]. However, core ASD symptoms may be the results of the effects of various genetic and environmental factors that may also affect organs other than central nervous system (CNS). Consequently, medical conditions affecting other organs may also affect ASD symptoms, partly through pain and discomfort. This makes it difficult to have reliable objective

diagnostic measures universally applicable for ASD children. This assumption is supported by the fact that ASD is characterized by multiple co-morbid medical conditions, with GI symptoms being the most common [3, 4].

Many co-morbid conditions reported in ASD subjects are associated with immune mediated inflammation in pathogenesis; GI symptoms found in ASD subjects have been in part implicated with chronic GI inflammation due to dysregulated gut immune responses to microbiota [5, 6]. Mounting evidence also indicates that many, but not all the ASD subjects show some immune abnormalities that affect almost every arm of the immune system [7, 8]. Moreover, given the fact that the immune system and the CNS interact closely interact [9], a role of neuroinflammation in ASD pathogenesis is highly suspected in a subset of ASD subjects.

In our previous studies, we focused on abnormalities of innate immunity, which plays a major role in the neuro-immune network including stress responses [10–12]. One of the reasons that we focused on innate immune abnormalities is based on findings from one of the most thoroughly studied animal models of ASD, maternal immune activation (MIA). MIA is induced by sterile stimulants of innate immunity in pregnant rodents [13]. MIA generates lasting effects on behavioral symptoms and the immune functions in offspring [13, 14]. It has been puzzling that how innate immunity, which lacks memory for specific antigens, can cause such lasting effects. However, discovery of innate immune memory (IIM) caused by initial stimuli through epigenetic regulations [15, 16], has helped us understand the lasting effects of dysregulated IIM in various inflammatory conditions. In fact, dysregulated IIM is now implicated in the pathogenesis of common neuropsychiatric diseases such as schizophrenia and depression [17, 18]. We found abnormalities of innate immunity in many, but not all the ASD subjects we studied by assessing cytokine profiles from purified monocytes [11].

Given both the considerable amount of abnormalities found in the monocyte cytokine profiles and the high frequency of co-morbid medical conditions in ASD subjects, questions understandably arise concerning the association of co-morbid conditions with changes in monocyte cytokine production. Many ASD subjects are treated with neurotropic medications including selective serotonin re-uptake inhibitors (SSRIs), anti-seizure medications used as mood stabilizers, neuroleptics, and medications for ADHD. These medications may also affect monocyte cytokine profiles. Therefore, this study addressed whether monocyte cytokine profiles differ depending on co-morbid conditions, ASD severity, and other clinical co-variables. The results indicate that co-morbid medical conditions are associated with changes in production of specific cytokines and such associated are not affected by other clinical co-variables.

#### **2. Materials and methods**

**Study subjects**: Study subjects were recruited following the study protocols (#17:53 and #19:53) approved by the institutional review. Signed consent forms were obtained prior to entering the study.

*ASD* subjects: ASD subjects (N = 109) were recruited from the Pediatric Allergy/Immunology Clinic at SPUH. Diagnosis of ASD was made at various autism diagnostic centers, including ours, based on the Autism Diagnostic Observation Scale (ADOS) and/or Autism Diagnostic Interview-Revisited (ADI-R), as well as other standard measures. ASD subjects were also evaluated for their behavioral symptoms and sleep habits with the Aberrant Behavior Checklist (ABC) [19] and the Children's Sleep Habits Questionnaires (CSHQ ) [20], respectively. Information regarding cognitive ability and adaptive skills were obtained from previous school

**85**

*W; Caucasian.*

**Table 1.**

results of CSHQ.

Age: Average ± SD (years) Median (Min to Max)

*Demographics of study subjects.*

*Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism…*

Demographics of study subjects were summarized in **Table 1**.

Antibody levels greater than 1.3 μg/ml were considered protective [26].

*Diagnosis of PANS like symptoms and sleep disorders:* Pediatric acute-onset neuropsychiatric syndrome (PANS) is a clinical diagnosis [27]. Most ASD subjects recruited to the study were diagnosed with PANS by other physicians. We attempted to validate the diagnosis based on the PANS diagnostic criteria [27]. In some ASD children, pre-existing neuropsychiatric symptoms made it difficult to apply the clinical diagnostic criteria of PANS. In this study, we categorized ASD patients with recurrent worsening behavioral symptoms following immune stimuli (typically microbial infection) more than 2×, not controlled by proper management of triggering stimuli as ASD subjects with PANS like symptoms. Diagnosis of sleep disorders that lasts at least more than 6 weeks is based on parental reports and

*Sample collection:* Venous blood samples were obtained by the physician in this study. We obtained one sample from each non-ASD controls. As for ASD subjects, we obtained multiple blood samples at different time points from select ASD subjects (N = *9*), in order to assess variability of *monocyte* cytokine profiles. If parents

> **ASD subjects (N = 109)**

Gender 95 M, 15 F (Female 13.8%) 19 M, 7 F (Female 26.9%)

Ethnicity 84 W, 5 AA, 19 Asian, 1 Mixed 22 W, 2 AA, 2 Mixed *Abbreviations used: AA; African American, ASD; autism spectrum disorders, SD; standard deviation,* 

12.4 ± 5.9 11.5 (2.2–26.3) **Non ASD controls (N = 26)**

> 12.2 ± 5.8 11.4 (3–28.8)

evaluation records performed within 1 year of enrollment in the study; these results were based on standard measures such as the Woodcock-Johnson III test (for cognitive ability), and Vineland Adaptive Behavior Scale (for adaptive skills) [21]. *Non-ASD controls:* A total of 26 non-ASD subjects served as controls. These subjects were recruited in the Pediatrics Subspecialty and General Pediatrics Clinics at our institution. These subjects were typically growing and satisfied our

*Diagnosis of food allergy (FA):* IgE mediated FA was diagnosed with reactions to offending food, by affecting the skin, GI, and/or respiratory tract immediately (within 2 hours) after intake with positive prick skin testing (PST) reactivity, and/or presence of food allergen-specific serum IgE. Non IgE mediated FA (NFA) was diagnosed if GI symptoms resolved, following implementation of a restricted diet (i.e., avoidance of offending food), and symptoms recurred with re-exposure to offending food [22]. NFA was also defined as being non-reactive to PFT and negative for serum

*Diagnosis of asthma and AR:* AR and allergic conjunctivitis (AC) were diagnosed when subjects had corresponding clinical features along with positive PST reactivity and/or positive serum IgE specific to causative allergens [23, 24]. Asthma was diagnosed following the asthma guidelines from the Expert Panel Report 3 [25]. *Diagnosis of Antibody deficiency syndrome:* When the subject revealed protective levels of antibodies in less than 11 of 14 serotypes of *Streptococcus pneumonia* after the booster dose of Pneumovax® or PCV13®, he/she was diagnosed with SAD [26].

*DOI: http://dx.doi.org/10.5772/intechopen.95548*

exclusion/inclusion criteria.

IgE specific for food allergens [22].

#### *Associations between Monocyte Cytokine Profiles and Co-Morbid Conditions in Autism… DOI: http://dx.doi.org/10.5772/intechopen.95548*

evaluation records performed within 1 year of enrollment in the study; these results were based on standard measures such as the Woodcock-Johnson III test (for cognitive ability), and Vineland Adaptive Behavior Scale (for adaptive skills) [21].

*Non-ASD controls:* A total of 26 non-ASD subjects served as controls. These subjects were recruited in the Pediatrics Subspecialty and General Pediatrics Clinics at our institution. These subjects were typically growing and satisfied our exclusion/inclusion criteria.

Demographics of study subjects were summarized in **Table 1**.

*Diagnosis of food allergy (FA):* IgE mediated FA was diagnosed with reactions to offending food, by affecting the skin, GI, and/or respiratory tract immediately (within 2 hours) after intake with positive prick skin testing (PST) reactivity, and/or presence of food allergen-specific serum IgE. Non IgE mediated FA (NFA) was diagnosed if GI symptoms resolved, following implementation of a restricted diet (i.e., avoidance of offending food), and symptoms recurred with re-exposure to offending food [22]. NFA was also defined as being non-reactive to PFT and negative for serum IgE specific for food allergens [22].

*Diagnosis of asthma and AR:* AR and allergic conjunctivitis (AC) were diagnosed when subjects had corresponding clinical features along with positive PST reactivity and/or positive serum IgE specific to causative allergens [23, 24]. Asthma was diagnosed following the asthma guidelines from the Expert Panel Report 3 [25].

*Diagnosis of Antibody deficiency syndrome:* When the subject revealed protective levels of antibodies in less than 11 of 14 serotypes of *Streptococcus pneumonia* after the booster dose of Pneumovax® or PCV13®, he/she was diagnosed with SAD [26]. Antibody levels greater than 1.3 μg/ml were considered protective [26].

*Diagnosis of PANS like symptoms and sleep disorders:* Pediatric acute-onset neuropsychiatric syndrome (PANS) is a clinical diagnosis [27]. Most ASD subjects recruited to the study were diagnosed with PANS by other physicians. We attempted to validate the diagnosis based on the PANS diagnostic criteria [27]. In some ASD children, pre-existing neuropsychiatric symptoms made it difficult to apply the clinical diagnostic criteria of PANS. In this study, we categorized ASD patients with recurrent worsening behavioral symptoms following immune stimuli (typically microbial infection) more than 2×, not controlled by proper management of triggering stimuli as ASD subjects with PANS like symptoms. Diagnosis of sleep disorders that lasts at least more than 6 weeks is based on parental reports and results of CSHQ.

*Sample collection:* Venous blood samples were obtained by the physician in this study. We obtained one sample from each non-ASD controls. As for ASD subjects, we obtained multiple blood samples at different time points from select ASD subjects (N = *9*), in order to assess variability of *monocyte* cytokine profiles. If parents

