Study of the Sex Differences in Inflammatory Diseases in Children

Study Overview
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Brief Summary
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Sexual differences in innate immune response have been demonstrated and were mainly attributed to the influence of the sex steroids (1-18). However, recent clinical data revealed significant differences in inflammatory markers between boys and girls suffering from acute and chronic inflammatory diseases (19-23). Sex hormone levels in prepubertal children are particularly low and insufficient to explain the gender differences observed in inflammatory conditions from neonates to the elderly, suggesting the contribution of another mechanism, such as the influence of genes situated on the sex chromosomes and involved in the inflammatory response. The aim of this work is to evaluate the role of the X chromosome in the sex differences in inflammatory diseases in children. In order to discriminate more precisely the role of the X chromosome relatively to the sex steroids in the sex-specific inflammatory response, some innate immune functions related to X-linked genes will be evaluated in whole blood from prepubertal children of both sexes, suffering from acute inflammatory processes such as pyelonephritis caused by Escherichia coli, pneumonia with pleural effusion caused by Streptococcus pneumoniae or sepsis

Detailed Description
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Many studies demonstrated immune differences between men and women suffering from acute and chronic inflammatory processes. In cases of acute inflammatory diseases, such as sepsis, females have better prognosis comparing to males (1,24-28). On the contrary, worse prognosis for women is observed in chronic inflammatory diseases such as asthma or cystic fibrosis (8-10,12,13,29). Sex-depended inflammatory response was attributed to the influence of sex hormones on the immune system. (2,15-18). However recent studies revealed differences in the clinical outcome but also in inflammatory markers between boys and girls suffering from acute and chronic inflammatory diseases (19-23). Sex hormone levels in prepubertal children are particularly low and insufficient to explain the gender differences observed in inflammatory conditions from neonates to the elderly, suggesting the contribution of another mechanism, such as the influence of genes situated on the sex chromosomes and involved in the inflammatory response. The aim of this work is to identify the potential X-linked mechanisms responsible for some of the differences between boys and girls in the inflammatory response, making the girls more at risk of developing complications in chronic inflammatory diseases and the boys more at risk of lethal complications in severe acute inflammatory diseases like sepsis. Several genes coding for innate immunity components are linked to the X chromosome such as diapedesis molecule CD99 or TLR pathway proteins genes. (30-33). X chromosome is also highly enriched in genes encoding micro RNAs (miRNAs) involved in the post-transcriptional regulation of gene expression which play a critical role in immune inflammatory response (34-36). Thus, in order to discriminate more precisely the role of the X chromosome relatively to the sex steroids in the sex-specific inflammatory response, some innate immune functions related to X-linked genes will be evaluated in whole blood from prepubertal children of both sexes, suffering from acute inflammatory processes such as pyelonephritis caused by Escherichia coli, pneumonia with pleural effusion caused by Streptococcus pneumoniae or sepsis. We will also study the correlations between inflammatory and clinical markers of the disease activity to identify prognosis indicators depending on the sex. Additionally, to delineate microbiome contribution, we will study the gut microbiota in stool samples obtained from the recruited patients.

Official Title
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Study of the Sex Differences in Inflammatory Diseases in Children

Conditions
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Sex Differences in Immune Response, Acute Inflammatory Diseases in Children

Intervention / Treatment
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* Other: Stool collection
* Other: Blood collection


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria of Experimental group : Male (XY) and female (XX) aged from 6 months to 7 years old. Subject hospitalized either for: (1) Urinary tract infection caused by Escherichia Coli, with: Temperature ≥ to 38,5°C Urinalysis Leukocyte esterase + AND/OR Nitrites + AND/OR pyuria (≥ 100WBC/mm³) AND/OR bacteriuria. Urinalysis Clean catch voided urine: > 10^4 Escherichia Coli colony form unit (CFU)/mm (urine collection method for children >3 years old or toilet trained children or by stimulation for children <3 years old) Transurethral bladder catheterisation: > 10^4 Escherichia Coli colony form unit (CFU)/mm³ (urine collection method for children <3 years old). Suprapubic aspiration: > 1 Escherichia Coli colony form unit (CFU)/mm³ (urine collection method for children <3 years old). (2) Pneumonia with pleural effusion with : Temperature ≥ 38,5°C Chest radiography/ultrasound: Pleural effusion Streptococcus pneumoniae identified on blood or pleural fluid culture or by PCR (3) Sepsis with: Documented or suspected infection Temperature < 36° or > 38.3°C Heart rhythm: 2 SD above normal for age 6-23 months: >180/min 24-71 months: >140/min 72-84 months: >130/min Respiratory Rate: 6-23 months: >35/min 24 - 71 months: >30/min 72-84 months: >20/min WBC: 6-23 months: >17500/µL or <5000/µL 24-71 months: >15500/µL or <6000/µL 72-84 months: >13500/µL or <4500/µL and/or CRP (blood) > 2SD above normal And at least two of the following: PaO2/FiO2 <300 Proven need for >50% FiO2 to maintain saturation ≥ 92% Need for mechanical ventilation Glasgow score < 11 Urine output < 0,5mL/kg/h for at least 2h Creatinine: 6-11 months: >0,4mg/dL 12-23 months: >0,5mg/dL 24-59 months: >0,8mg/dL 60-84 months: >1mg/dL Or creatinine increase more than 0,5 mg/dL Platelet count <100000/mL Bilirubin >2 mg/dL Mean arterial pressure (MAP) 6-11 months: <55 mmHg 12 -23 months: <60 mmHg 24-59 months: <62 mmHg 60-84 months: <65 mmHg SBP less than two SD below normal for age Prolonged capillary refill: > 5 sec Inclusion Criteria of Control group : Male (XY) and female (XX) aged from 6 months to 7 years old. Scheduled surgical intervention for a non-infectious pathology. Exclusion Criteria: Use of antithrombotic drugs (acetylsalicylic acid, thienopyridines, dipyridamol, glycoprotein IIb / IIIa antagonists, vitamin K antagonists, heparins). Congenital or acquired immunodeficiency: immunosuppressive drugs, hematopoietic stem cells transplantation, immunoglobulin therapy, extracorporeal membrane oxygenation (ECMO). Hemodialysis. 48h following cardiac operation of any type. Malignant cancer. HIV.

Ages Eligible for Study
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Minimum Age: 6 Months
Maximum Age: 7 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Non-Randomized
Intervention Model: Parallel Assignment
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Children suffering from acute inflammatory processes.<br>The study population will consist of male and female children, aged from 6 months to 7 years old, admitted to the hospital for one of the three following types of acute inflammatory processes: Urinary tract infection caused by Escherichia coli Pneumonia with pleural effusion caused by Streptococcus pneumoniae Sepsis | Other: Blood collection<br>* Blood samples collections to evaluation of the potential role of the sex chromosomes in the innate immune response by analyzing inflammatory cytokine production (IL-1β, IL-6, IL-8, IL-10, TNF-α and IFN-α), studying the cell diapedesis receptor CD99 on PMNs, monocytes, and lymphocytes, analyzing the contribution of X-linked genes of the TLR pathways and the influence of X-linked miRNAs.<br>Other: Stool collection<br>* Fecal sample collection to delineate microbiome contribution, we will study the gut microbiota in faecal samples obtained from the recruited patients.<br>|
| Other: Control group<br>Male and female children, aged from 6 months to 7 years old, admitted to the hospital for a scheduled operation for a non-inflammatory pathology. | Other: Blood collection<br>* Blood samples collections to evaluation of the potential role of the sex chromosomes in the innate immune response by analyzing inflammatory cytokine production (IL-1β, IL-6, IL-8, IL-10, TNF-α and IFN-α), studying the cell diapedesis receptor CD99 on PMNs, monocytes, and lymphocytes, analyzing the contribution of X-linked genes of the TLR pathways and the influence of X-linked miRNAs.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Whole blood production of cytokine IL-6 | The production of IL6 is measured by multiplex techniques. | within 24 hours of hospital admission (Day 0) | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Whole blood production of cytokine IL-1β |  | within 24 hours of hospital admission (Day 0) | 
| Whole blood production of cytokine IL-8 |  | within 24 hours of hospital admission (Day 0) | 
| Whole blood production of cytokine IL-10 |  | within 24 hours of hospital admission (Day 0) | 
| Whole blood production of cytokine TNF-α |  | within 24 hours of hospital admission (Day 0) | 
| Whole blood production of cytokine interferon-α |  | within 24 hours of hospital admission (Day 0) | 
| Intracellular quantity of the phosphorylated forms of NF-κB p65 in leukocyte population. |  | within 24 hours of hospital admission (Day 0) | 
| Intracellular quantity of the phosphorylated forms of ERK1/2 in leukocyte population. |  | within 24 hours of hospital admission (Day 0) | 
| Intracellular quantity of the phosphorylated forms of p38 MAPK in leukocyte population. |  | within 24 hours of hospital admission (Day 0) | 
| Expression of the cell diapedesis receptor CD99 on PMNs | Measurements of cell diapedesis receptor CD99 on leukocytes will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of the cell diapedesis receptor CD99 on monocytes | Measurements of cell diapedesis receptor CD99 on leukocytes will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of the cell diapedesis receptor CD99 on lymphocytes | Measurements of cell diapedesis receptor CD99 on leukocytes will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of TLR2 on PMNs | Measurements of intracellular phosphorylated forms of TLR pathway proteins as well as the expression of TLR2 and TLR4 will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of TLR2 on monocytes | Measurements of intracellular phosphorylated forms of TLR pathway proteins as well as the expression of TLR2 and TLR4 will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of TLR2 on lymphocytes | Measurements of intracellular phosphorylated forms of TLR pathway proteins as well as the expression of TLR2 and TLR4 will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of TLR4 on PMNs | Measurements of intracellular phosphorylated forms of TLR pathway proteins as well as the expression of TLR2 and TLR4 will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of TLR4 on monocytes | Measurements of intracellular phosphorylated forms of TLR pathway proteins as well as the expression of TLR2 and TLR4 will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| Expression of TLR4 on lymphocytes | Measurements of intracellular phosphorylated forms of TLR pathway proteins as well as the expression of TLR2 and TLR4 will be performed by flow cytometry | within 24 hours of hospital admission (Day 0) | 
| BTK gene expression | Measurements will be performed using the Quantitect Reverse Transcription Kit (Qiagen, Manchester, UK) for quantitative PCR (qPCR) on leucocytes. | within 24 hours of hospital admission (Day 0) | 
| IRAK1 gene expression | Measurements will be performed using the Quantitect Reverse Transcription Kit (Qiagen, Manchester, UK) for quantitative PCR (qPCR) on leucocytes. | within 24 hours of hospital admission (Day 0) | 
| NEMO gene expression | Measurements will be performed using the Quantitect Reverse Transcription Kit (Qiagen, Manchester, UK) for quantitative PCR (qPCR) on leucocytes. | within 24 hours of hospital admission (Day 0) | 
| Expression of X-linked miRNAs in leucocytes | Expression of X-linked miRNAs is measured by sequencing and qRT-PCR on leucocytes and/or plasma samples. | within 24 hours of hospital admission (Day 0) | 
| Expression of X-linked miRNAs in plasma | Expression of X-linked miRNAs is measured by sequencing and qRT-PCR on leucocytes and/or plasma samples. | within 24 hours of hospital admission (Day 0) | 
| Leukocyte population | White blood cell count including neutrophils, monocytes, monocytes subtypes and lymphocytes. | within 24 hours of hospital admission (Day 0) | 
| Leukocyte population | White blood cell count including neutrophils, monocytes, monocytes subtypes and lymphocytes. Only applicable for the sepsis sub-group | Day 1 | 
| Leukocyte population | White blood cell count including neutrophils, monocytes, monocytes subtypes and lymphocytes. Only applicable for the sepsis sub-group | Day 2 | 
| Leukocyte population | White blood cell count including neutrophils, monocytes, monocytes subtypes and lymphocytes. Only applicable for the sepsis sub-group | Day 3 | 
| CRP |  | within 24 hours of hospital admission (Day 0) | 
| CRP | Only applicable for the sepsis sub-group | Day 1 | 
| CRP | Only applicable for the sepsis sub-group | Day 2 | 
| CRP | Only applicable for the sepsis sub-group | Day 3 | 
| Total 17β-estradiol |  | within 24 hours of hospital admission (Day 0) | 
| Testosterone |  | within 24 hours of hospital admission (Day 0) | 
| IGF1 |  | within 24 hours of hospital admission (Day 0) | 
| Microbiome analysis |  | During subject hospitalisation | 
| pSOFA score | Only applicable for the sepsis sub-group. The pSOFA will be evaluated every 24 hours in order to compare laboratory and clinical data. The score will be based on the PaO2: FiO2 or SpO2: FiO2 ratio, the platelet count, the bilirubin level, the Mean Arterial Pressure (MAP), the Glasgow score and the creatinine level. | within 24 hours of hospital admission (Day 0) | 
| pSOFA score | Only applicable for the sepsis sub-group. The pSOFA will be evaluated every 24 hours in order to compare laboratory and clinical data. The score will be based on the PaO2: FiO2 or SpO2: FiO2 ratio, the platelet count, the bilirubin level, the Mean Arterial Pressure (MAP), the Glasgow score and the creatinine level. | Day 1 | 
| pSOFA score | Only applicable for the sepsis sub-group. The pSOFA will be evaluated every 24 hours in order to compare laboratory and clinical data. The score will be based on the PaO2: FiO2 or SpO2: FiO2 ratio, the platelet count, the bilirubin level, the Mean Arterial Pressure (MAP), the Glasgow score and the creatinine level. | Day 2 | 
| pSOFA score | Only applicable for the sepsis sub-group. The pSOFA will be evaluated every 24 hours in order to compare laboratory and clinical data. The score will be based on the PaO2: FiO2 or SpO2: FiO2 ratio, the platelet count, the bilirubin level, the Mean Arterial Pressure (MAP), the Glasgow score and the creatinine level. | Day 3 | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Sex, immunology, inflammation, child, sepsis, UTI, pneumonia