**Table 1.**

*Demographics of study subjects.*

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

conditions, with GI symptoms being the most common [3, 4].

diagnostic measures universally applicable for ASD children. This assumption is supported by the fact that ASD is characterized by multiple co-morbid medical

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 neuroinflamma-

tion 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

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

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

*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

**84**

purified monocytes [11].

clinical co-variables.

**2. Materials and methods**

were obtained prior to entering the study.

or study subjects preferred, we applied a topical lidocaine/prilocaine cream (Emla cream®) to the site of venipuncture prior to blood sampling.

**Cell cultures:** Ficoll–Hypaque density gradient centrifugation was used for separating PBMCs. From PBMCs, PBMo were further purified using a column of magnetic beads labeled with anti-CD3, CD7, CD16, CD19, CD56, CD123, and glycophorin A (monocyte separation kit II – human, MILTENYI BIOTEC, Cambridge, MA, United States). Combination of these antibodies depletes T, B, natural killer, and dendritic cells from PBMCs.

Cytokine production by purified PBMo was induced by incubating cells overnight (2.5 × 105 cells/ml) with a panel of agonists of TLRs. This assay system was designed to reflect the effects of microbial byproducts commonly encountered in real life. Lipopolysaccharide (LPS), a TLR4 agonist, represents a signaling pathway activated in response to a gram negative [G (−)] bacteria. Zymosan, a TLR2/6 agonist, mimics an innate activation signal in response to G (+) bacteria and fungi. CL097, a TLR7/8 agonist, activates innate signaling pathways in response to ssRNA viruses that cause common respiratory infection. Candida heat extract as a source of ß-glucan, a dectin-1 agonist, was used as well as a C-lectin receptor agonist. PBMos were incubated overnight with LPS (0.1 μg/ml, GIBCO-BRL, Gaithersburg, MD, USA), zymosan (50 μg/ml, Sigma-Aldrich, St. Luis, Mo), C097 (water-soluble derivative of imidazoquinoline, 20 μM, InvivoGen, San Diego, CA, USA), and candida heat extract (HCKA, heat killed *Candida albicans* (107 cells/ml, InVivogen, San Diego, CA) in RPMI 1640 with additives as previously described [28]. Overnight incubation (16-20 h) was adequate to induce the optimal responses in this setting in previous studies [11]. Cytokine levels in the culture supernatant were then measured.

Levels of CCL2, IL-1β, IL-6, IL-10, IL-12p40, transforming growth factor-ß (TGF-ß), tumor TNF-α, and sTNFRII cytokines were measured by enzyme-linked immuno-sorbent assay (ELISA); 10–100 μl/well supernatants were used for ELISA. The OptEIA™ Reagent Sets (BD Biosciences, San Jose, CA, USA) were used for ELISA of IL-1ß, IL-6, IL-10, IL-12p40, and TNF-α. For CCL2, sTNFRII, and TGF-ß ELISA, reagents were obtained from BD Biosciences and R & D (Minneapolis, MN, USA). IL-23 ELISA kit was purchased from eBiosciences, San Diego, CA. Intra- and inter-variations of cytokine levels were less than 5%.

**Statistical analysis**: We used a two tailed Mann–Whitney test for comparison of two sets of numerical data. Kruskal-Wallis test was used for comparison of more than 2 sets of numerical data. When assessing differences in frequency between two groups, we used the Fisher exact test. For assessing differences in frequency among multiple groups, we used the Chi-square test and the Likelihood ratio. P value of less than 0.05 was considered nominally significant. Co-variance analysis was done with the use of analysis of variance (ANOVA) for a fixed factor or for a variable factor. NCSS2020 (NCSS, LLC. Kaysville, UT) was used for such statistical analysis.

#### **3. Results**

**Clinical characteristics:** Frequencies of co-morbid conditions among the recruited ASD subjects are summarized in **Table 2**. These results are consistent with the results of our previous studies [12, 29]. Age and gender were not associated with ASD severity (data now shown). Frequencies of co-morbid conditions and the use of neurotropic medications did not differ due to ASD severity in 108 ASD subjects who were verified ASD severity (**Table 3**).

**Changes in monocyte cytokine production depending on ASD severity**: We then examined whether monocyte cytokine profiles differed with ASD severity

**87**

(**Table 4**).

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

**Comorbid conditions ASD subjects (N = 109) Controls (N = 26)**

 symptoms 71/109 (65.1%) 0 history of NFA 68/109 (63.4%) 2/26 (7.7%) Seizure disorders 18/109 (16.5%) 0 Asthma 5/109 (4.6%) 0 Allergic rhinitis 17/109 (15.6%) 0 Specific antibody deficiency 26/109 (23.9%) 0 PANS like symptoms 62/109 (56.9%) 0 Disturbed Sleep 48/109 (44.0%) 0

*Abbreviations used: ASD; autism spectrum disorder, GI; gastrointestinal, NFA; non-IgE mediated food allergy,* 

 **severity Level 1 Level 2 Level 3 Chi-Square**

GI symptoms 14/27<sup>2</sup> 20/32 36/49 p > 0.1 Seizure disorder 1/27 6/32 10/49 p = 0.081 Antibody deficiency 6/27 10/32 10/49 p > 0.1 PANS like symptoms 12/27 19/32 30/49 p > 0.1 Disturbed sleep 8/27 16/32 23/49 p > 0.1

SSRIs 6/27 8/32 4/49 p > 0.1 Anti-seizure medications 3/27 9/32 8/49 p > 0.1 ADHD medications 3/27 3/32 3/49 p > 0.1 Neuroleptics 4/27 1/32 9/49 p = 0.079

*Abbreviations used: ASD; autism spectrum disorder, GI; gastrointestinal, NFA; non-IgE mediated food allergy, ADHD: attention deficiency hyperactivity disorder, PANS; pediatric acute-onset neuropsychiatric syndrome, SSRI:* 

*One ASD subject was excluded from this analysis due to lack of validation of ASD severity.*

*Frequencies of Co-morbid conditions and medication use did not differ due to ASD severity.*

and if such changes were affected by other clinical co-variables. ASD severity was shown to be associated with changes in production of TNF-α, IL-1ß, IL-10, and CCL2, and TNF-α/sTNFRII ratios (**Table 4**). However, production of inflammatory monocyte cytokines (TNF-α and IL-1ß) under several culture conditions is affected by presence of co-morbid conditions and the use of ADHD medications

**Changes in monocyte cytokine production depending on co-morbid conditions:** Since associations between ASD severity and monocyte cytokine profiles were often affected by other co-morbid conditions and medication use (**Table 4**), we also evaluated whether changes in monocyte cytokine profiles in ASD

*Co-morbid conditions with objective diagnostic measures:* We evaluated changes in monocyte cytokine profiles in association with co-morbid conditions which were

subjects were affected by the presence of co-morbid conditions.

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

*PANS; pediatric acute-onset neuropsychiatric syndrome.*

*Frequency of comorbid conditions in the study subjects.*

GI1

*1*

**Table 2.**

**ASD1**

**Medications**

*selective serotonin reuptake inhibitor.*

*1*

*2*

**Table 3.**

**Co-morbid conditions**

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


*1 Abbreviations used: ASD; autism spectrum disorder, GI; gastrointestinal, NFA; non-IgE mediated food allergy, PANS; pediatric acute-onset neuropsychiatric syndrome.*

#### **Table 2.**

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

cream®) to the site of venipuncture prior to blood sampling.

dida heat extract (HCKA, heat killed *Candida albicans* (107

inter-variations of cytokine levels were less than 5%.

who were verified ASD severity (**Table 3**).

and dendritic cells from PBMCs.

night (2.5 × 105

measured.

or study subjects preferred, we applied a topical lidocaine/prilocaine cream (Emla

**Cell cultures:** Ficoll–Hypaque density gradient centrifugation was used for separating PBMCs. From PBMCs, PBMo were further purified using a column of magnetic beads labeled with anti-CD3, CD7, CD16, CD19, CD56, CD123, and glycophorin A (monocyte separation kit II – human, MILTENYI BIOTEC, Cambridge, MA, United States). Combination of these antibodies depletes T, B, natural killer,

Cytokine production by purified PBMo was induced by incubating cells over-

designed to reflect the effects of microbial byproducts commonly encountered in real life. Lipopolysaccharide (LPS), a TLR4 agonist, represents a signaling pathway activated in response to a gram negative [G (−)] bacteria. Zymosan, a TLR2/6 agonist, mimics an innate activation signal in response to G (+) bacteria and fungi. CL097, a TLR7/8 agonist, activates innate signaling pathways in response to ssRNA viruses that cause common respiratory infection. Candida heat extract as a source of ß-glucan, a dectin-1 agonist, was used as well as a C-lectin receptor agonist. PBMos were incubated overnight with LPS (0.1 μg/ml, GIBCO-BRL, Gaithersburg, MD, USA), zymosan (50 μg/ml, Sigma-Aldrich, St. Luis, Mo), C097 (water-soluble derivative of imidazoquinoline, 20 μM, InvivoGen, San Diego, CA, USA), and can-

Diego, CA) in RPMI 1640 with additives as previously described [28]. Overnight incubation (16-20 h) was adequate to induce the optimal responses in this setting in previous studies [11]. Cytokine levels in the culture supernatant were then

Levels of CCL2, IL-1β, IL-6, IL-10, IL-12p40, transforming growth factor-ß (TGF-ß), tumor TNF-α, and sTNFRII cytokines were measured by enzyme-linked immuno-sorbent assay (ELISA); 10–100 μl/well supernatants were used for ELISA. The OptEIA™ Reagent Sets (BD Biosciences, San Jose, CA, USA) were used for ELISA of IL-1ß, IL-6, IL-10, IL-12p40, and TNF-α. For CCL2, sTNFRII, and TGF-ß ELISA, reagents were obtained from BD Biosciences and R & D (Minneapolis, MN, USA). IL-23 ELISA kit was purchased from eBiosciences, San Diego, CA. Intra- and

**Statistical analysis**: We used a two tailed Mann–Whitney test for comparison of two sets of numerical data. Kruskal-Wallis test was used for comparison of more than 2 sets of numerical data. When assessing differences in frequency between two groups, we used the Fisher exact test. For assessing differences in frequency among multiple groups, we used the Chi-square test and the Likelihood ratio. P value of less than 0.05 was considered nominally significant. Co-variance analysis was done with the use of analysis of variance (ANOVA) for a fixed factor or for a variable factor. NCSS2020 (NCSS, LLC. Kaysville, UT) was used for such statistical analysis.

**Clinical characteristics:** Frequencies of co-morbid conditions among the recruited ASD subjects are summarized in **Table 2**. These results are consistent with the results of our previous studies [12, 29]. Age and gender were not associated with ASD severity (data now shown). Frequencies of co-morbid conditions and the use of neurotropic medications did not differ due to ASD severity in 108 ASD subjects

**Changes in monocyte cytokine production depending on ASD severity**: We then examined whether monocyte cytokine profiles differed with ASD severity

cells/ml) with a panel of agonists of TLRs. This assay system was

cells/ml, InVivogen, San

**86**

**3. Results**

*Frequency of comorbid conditions in the study subjects.*


*1 Abbreviations used: ASD; autism spectrum disorder, GI; gastrointestinal, NFA; non-IgE mediated food allergy, ADHD: attention deficiency hyperactivity disorder, PANS; pediatric acute-onset neuropsychiatric syndrome, SSRI: selective serotonin reuptake inhibitor.*

*2 One ASD subject was excluded from this analysis due to lack of validation of ASD severity.*

#### **Table 3.**

*Frequencies of Co-morbid conditions and medication use did not differ due to ASD severity.*

and if such changes were affected by other clinical co-variables. ASD severity was shown to be associated with changes in production of TNF-α, IL-1ß, IL-10, and CCL2, and TNF-α/sTNFRII ratios (**Table 4**). However, production of inflammatory monocyte cytokines (TNF-α and IL-1ß) under several culture conditions is affected by presence of co-morbid conditions and the use of ADHD medications (**Table 4**).

**Changes in monocyte cytokine production depending on co-morbid conditions:** Since associations between ASD severity and monocyte cytokine profiles were often affected by other co-morbid conditions and medication use (**Table 4**), we also evaluated whether changes in monocyte cytokine profiles in ASD subjects were affected by the presence of co-morbid conditions.

*Co-morbid conditions with objective diagnostic measures:* We evaluated changes in monocyte cytokine profiles in association with co-morbid conditions which were


*1 ANOVA co-variance analysis revealed an association with the use of ADHD medications (p < 0.02 and p < 0.05 under culture conditions stimulated with CL097 and ß-glucan+LPS, respectively).*

*2 ANOVA co-variance analysis revealed an association with GI symptoms (p < 0.05).*

*3 ANOVA co-variance analysis revealed an association with PANS like symptoms (p < 0.05).*

*4 ANOVA co-variance analysis revealed an association with Disturbed sleep (p < 0.05).*

*5 Abbreviations used: CCL2; C-C chemokine ligand 2, IL; interleukin, LPS; lipopolysaccharide, TNF; tumor necrosis factor.*

#### **Table 4.**

*Differences in monocyte cytokine production depending on ASD severity.*

evaluated with objective measures as defined in the method section. We found that such co-morbid conditions were observed in more than 15% of our ASD study subjects; these include GI symptoms, seizure disorders, AR, and SAD. Our results revealed that there are significant associations between monocyte cytokine production and ASD co-morbid conditions as described above (**Table 5**). Presence of GI symptoms are mainly associated with changes in TNF-α production and the ratio of TNF-α/sTNFRII ratios under several culture conditions. Co-variance analysis showed that these parameters were mostly not affected by other clinical co-variables including medication use. The exceptions are TNF-α production and TNF-α/ sTNFRII ratios under zymosan stimulated cultures, which are affected by ASD severity (**Table 5**). Diagnosis of seizure disorders was the most notably associated with changes in IL-1ß production and IL-1ß/IL-10 ratios under the CL097 stimulated cultures, independent of clinical co-variables that we assessed (**Table 5**). The AR diagnosis is mainly associated with changes in sTNFRII production. The SAD diagnosis is mainly associated with changes in IL-6 and IL-10 production. Most of these cytokine parameters were again not affected by the other clinical variables that we assessed.

*Co-morbid conditions based on clinical diagnosis:* Although assessment of PANS like symptoms and sleep disorder were diagnosed without objective measures, given the high frequency of these conditions, we also assessed differences in monocyte cytokine parameters in association with these two co-morbid conditions. Significant differences in certain monocyte cytokine parameters were found in the presence of PANS like symptoms and sleep disorders (**Table 6**). PANS like conditions were associated with changes in inflammatory cytokines (IL-6 and IL-1ß), as well as sTNFRII and CCL2. Only IL-1ß production was affected by other clinical covariables. As for sleep disorders, changes in TGF-ß levels were mainly associated with the presence of sleep disorders and changes in TGF-ß productions was independent of clinical co-variables.

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**Comorbid conditions**

**GI symptoms**

TNF-α (LPS) TNF-α (zymosan)2

TNF-α (ß-glucan)

TNF-α/sTNFRII (LPS)

TNF-α/sTNFRII (zymosan)2

TNF-α/sTNFRII (ß-glucan)

**Seizure disorders**

IL-1ß (CL097) IL-1ß/IL-10 (CL097)

TNF-α/sTNFRII

(CL097) CCL2 (CL097) IL-1ß (ß-glucan)

**Allergic rhinitis**

sTNFRII (LPS)

IL-1ß (CL097)2

sTNFRII (ß-glucan)2

sTNFRII (LPS + ß-glucan)

**Antibody deficiency**

IL-6 (medium)

IL-6 (LPS) IL-6 (zymosan)2

**ASD1 with comorbid condition**

**N = 81** 685.1 ± 744.83 1498.8 ± 1055.6 1961.6 ± 1108.2

0.69 ± 0.89 2.98 ± 2.61 6.84 ± 6.79

**N = 24** 3732.3 ± 1092.8

5.10 ± 7.98 3.89 ± 6.74 18862 ± 19577 1955.9 ± 996.9

**N = 20** 1538.1 ± 395.5 5038.3 ± 1368.3

497.8 ± 225.2 488.8 ± 249.4

**N = 31** 2303.5 ± 1935.8

19536 ± 9625 5510.5 ± 2085.6

**ASD without comorbid condition**

**N = 42** 373.0 ± 366.5 1047.6 ± 709.6 1609.7 ± 1135.7

0.50 ± 1.27 2.67 ± 5.68 3.88 ± 2.31

**N = 99** 4566.4 ± 1357.5

8.98 ± 12.44 8.04 ± 13.74 14588 ± 9672 2546.4 ± 972.1

**N = 103** 1284.0 ± 493.7 4285.8 ± 1315.6

414.1 ± 286.6 392.5 ± 297.3

**N = 92** 3869.2 ± 2581.629553 ± 18279

6309.2 ± 1974.7

388.8 ± 380.0

**Non-ASD Control**

**N = 26** 474.4 ± 505.8 1609.7 ± 748.3 1931.1 ± 948.5

1.28 ± 3.53 3.54 ± 3.61 13.5 ± 18.0

**N = 26** 3715.5 ± 1367.9

3.98 ± 2.53 4.31 ± 3.48 11499 ± 7621 21767.0 ± 999.9

**N = 26**

1172.5 ± 504.2 3715.5 ± 1357.9

340.8 ± 257.7 371.6 ± 354.3

**N = 26**

3373.3 ± 1562.9 20152 ± 12909 7538.5 ± 9310.0

p < 0.01

p < 0.01

p = 0.106

p < 0.05

p < 0.01

p < 0.05

p = 0.07179

p < 0.01 p < 0.001

p < 0.02

p < 0.02

p = 0.056

p < 0.01 p < 0.01 p = 0.113 p < 0.05 p < 0.05 p < 0.01

**Kruskal-Wallis test**

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

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


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

*Autism Spectrum Disorder - Profile, Heterogeneity, Neurobiology and Intervention*

 **severity Level 1**

**ASD severity Level 2**

**N = 33 N = 37 N = 52**

3.78 ± 3.34 8.43 ± 10.43 8.73 ± 17.27 p < 0.05

TNF-α (CLO97)1 2390.2 ± 1786.9 2720.2 ± 983.3 2073.0 ± 1171.6 p < 0.01 IL-10 (CLO97) 1318.1 ± 595.3 918.2 ± 576.1 1009.9 ± 607.2 p < 0.05

TNF-α (ß-glucan)3 1816.2 ± 1139.4 2360.4 ± 1249.9 1512.2 ± 883.9 p < 0.005 IL-1ß (ß-glucan)4 2578.7 ± 922.0 2809.7 ± 984.4 2062.9 ± 949.7 p < 0.005 TNF-α (ß-glucan+LPS)1 2338.0 ± 259.0 2658.4 ± 984.5 2000.2 ± 916.5 p < 0.01 CCL2 (ß-glucan+LPS) 2708.0 ± 2477.4 1758.6 ± 16681 1574.8 ± 1553.1 p < 0.05 CCL2 (zymosan) 9105.2 ± 6631.1 7668.4 ± 4973.2 5870.3 ± 4414.4 p < 0.05

*ANOVA co-variance analysis revealed an association with the use of ADHD medications (p < 0.02 and p < 0.05* 

*Abbreviations used: CCL2; C-C chemokine ligand 2, IL; interleukin, LPS; lipopolysaccharide, TNF; tumor necrosis* 

**ASD severity Level 3**

**Kruskal-Wallis test**

**ASD5**

*under culture conditions stimulated with CL097 and ß-glucan+LPS, respectively).*

*Differences in monocyte cytokine production depending on ASD severity.*

*ANOVA co-variance analysis revealed an association with GI symptoms (p < 0.05).*

*ANOVA co-variance analysis revealed an association with Disturbed sleep (p < 0.05).*

*ANOVA co-variance analysis revealed an association with PANS like symptoms (p < 0.05).*

**Monocyte cytokine production**

TNF-α/sTNFRII (CLO97)2

*1*

*2*

*3*

*4*

*5*

*factor.*

**Table 4.**

evaluated with objective measures as defined in the method section. We found that such co-morbid conditions were observed in more than 15% of our ASD study subjects; these include GI symptoms, seizure disorders, AR, and SAD. Our results revealed that there are significant associations between monocyte cytokine production and ASD co-morbid conditions as described above (**Table 5**). Presence of GI symptoms are mainly associated with changes in TNF-α production and the ratio of TNF-α/sTNFRII ratios under several culture conditions. Co-variance analysis showed that these parameters were mostly not affected by other clinical co-variables including medication use. The exceptions are TNF-α production and TNF-α/ sTNFRII ratios under zymosan stimulated cultures, which are affected by ASD severity (**Table 5**). Diagnosis of seizure disorders was the most notably associated with changes in IL-1ß production and IL-1ß/IL-10 ratios under the CL097 stimulated cultures, independent of clinical co-variables that we assessed (**Table 5**). The AR diagnosis is mainly associated with changes in sTNFRII production. The SAD diagnosis is mainly associated with changes in IL-6 and IL-10 production. Most of these cytokine parameters were again not affected by the other clinical variables

*Co-morbid conditions based on clinical diagnosis:* Although assessment of PANS like symptoms and sleep disorder were diagnosed without objective measures, given the high frequency of these conditions, we also assessed differences in monocyte cytokine parameters in association with these two co-morbid conditions. Significant differences in certain monocyte cytokine parameters were found in the presence of PANS like symptoms and sleep disorders (**Table 6**). PANS like conditions were associated with changes in inflammatory cytokines (IL-6 and IL-1ß), as well as sTNFRII and CCL2. Only IL-1ß production was affected by other clinical covariables. As for sleep disorders, changes in TGF-ß levels were mainly associated with the presence of sleep disorders and changes in TGF-ß productions

**88**

that we assessed.

was independent of clinical co-variables.


*with allergic rhinitis was affected with the use of anti-seizure medications (p < 0.05). Changes in production of IL-6 (zymosan) and IL-10 production (LPS) with antibody deficiency was affected with the use of neuroleptics/SSRIs and PANS like symptoms.*

*3The results were expressed as a mean ± SD. Cytokine levels were shown as pg/ml.*
