Evaluation Of Serum HMGB1 And TLR4 As Potential Targets For Predicting And Treating Infantile Epileptic Spasms In Children Receiving ACTH Therapy

Objectives:1.To investigate the presence of immune dysregulation in Infantile epileptic spasms syndrome2.To explore the immunomodulatory pathways involved in the treatment of Infantile epileptic spasms syndrome using adrenocorticotropic hormone(ACTH).3.To identify potential biomarkers associated with the effectiveness of ACTH therapy.Methods:1.A total of 70 blood samples were collected from 35 infants diagnosed with Infantile epileptic spasms syndrome at Anhui Children’s Hospital,both before and after treatment.An additional 35 blood samples were collected from healthy control children.2.Enzyme-linked immunosorbent assay(ELISA)was employed to measure the serum levels of high-mobility group-box protein 1(HMGB1),Toll-like receptor 4(TLR4),and tumor necrosis factor-alpha(TNF-α).3.Flow cytometry was utilized to analyze the percentages of T lymphocyte subsets(CD3+,CD4+,CD8+),B lymphocyte subset(CD3-CD19+B),and natural killer cells(CD3-CD16+56+NK)in the blood.4.Chemiluminescent immunoassay was conducted to measure the levels of interleukin1β(IL-1β),interleukin-2 receptor(IL-2R),interleukin-6(IL-6),interleukin-8(IL-8),and interleukin-10(IL-10)in the serum.5.The burden of amplitudes and epileptiform discharges score(BASED)was used to assess the degree of EEG dysrhythmia in the infants.6.The Gesell scale was employed to evaluate the neurodevelopmental status of the infants.7.Independent sample t-tests and chi-square tests were employed to evaluate the differences in demographic characteristics and levels of immune-inflammatory factors between the case group and the control group.Paired t-tests were used to assess the differences in immune-inflammatory factor levels before and after treatment.Independent sample t-tests were conducted to compare the distribution differences among different efficacy groups.Binary logistic regression analysis,adjusting for baseline characteristics such as age,gender,medical history,family history,and disease history,was used to evaluate the relationship between immune-inflammatory factor differences and the effectiveness of ACTH.Results:1.Comparison of immune indexes between the case group and the control group before treatment:The result revealed that in the case group,the levels of HMGB1[(1368.63±815.44)pg/ml],TLR4[(0.82±0.61)ng/ml],IL-1β[(18.63±37.29)pg/ml],IL2R[(1425.97±37.29)U/ml],IL-6[(8.10±19.85)U/ml]were significantly higher compared to the healthy control group[(p<0.05)].Furthermore,the case group[(p<0.05)]exhibited significantly lower percentages of CD3+T lymphocytes[(56.12±9.76)%],CD3+CD4+T lymphocytes[(35.46±7.27)%],and the case group[(p<0.05)]exhibited significantly higher percentages of CD3-CD19+B lymphocytes[(35.21±11.18)%].2.Comparison of immune indexes between pre-therapy and post-therapy:After 2 weeks of ACTH treatment,significant changes were observed in TLR4[(0.60 ±0.42)ng/ml],IL-1β[(3.51±5.38)pg/ml],IL-2R[(869.63±476.80)U/ml],the percentages of CD3+T lymphocytes[(62.24 ± 8.61)%],CD3+CD4+T lymphocytes[(40.40 ± 7.27)%],and CD3-CD16+56+NK cells[(5.85±2.80)%]as well.These changes suggest a reduction in TLR4,IL-1β,IL-2R,as well as an increase in the percentages of CD3+T lymphocytes and CD3+CD4+T lymphocytes after treatment(p<0.05).However,the remaining parameters did not exhibit significant changes(p<0.05).3.Comparison of immune indexes between the effective group(n=19)and the ineffective group(n=16)of ACTH treatment.:Standardized Z-scores of the indicator data were calculated by subtracting the post-treatment values from the pre-treatment values.The Z-scores of serum HMGB1(-0.70±0.77)and TLR4(-0.55±0.97)in the effective group were significantly lower than those in the ineffective group,which exhibited serum HMGB1(0.83±0.46)and TLR4(0.65±0.56)Z-scores(p<0.05).Binary logistic regression analysis,adjusted for baseline characteristics such as age,gender,medical history,family history,and disease history,revealed a negative multiplicative interaction between the expression of HMGB1(OR=791.08,95%CI 2.72-229700.49,p=0.021)andTLR4(OR=19.33,95%CI 1.64-227.89,p=0.019)in the serum of patients with Infantile epileptic spasms syndrome and the effectiveness of ACTH4.Clinical outcomes:Out of the 35 patients,16 cases(45.71%)showed an ineffective response to ACTH treatment(i.e.,less than 50%reduction in seizure frequency).One the other hand,12 cases(34.29%)achieved complete remission.The effective rate,defined as a reduction in seizure frequency of 50%or more,was observed in 19 cases(54.29%).At baseline,all 35 patients had a Burden of Amplitudes and Epileptiform Discharges(BASED)rating of ≥ 4(100%),indicating highly dysrhythmic EEG patterns.Unilateral epileptiform discharges were present in 42.86%(15/35)of cases,while bilateral discharges were observed in 57.14%(20/35)of cases.Focal epileptiform discharges accounted for 45.71%(16/35)of cases,and generalized discharges accounted for 54.29%(19/35)of cases.After 2 weeks of ACTH treatment,EEG improvement(rating≤2)was observed in 20 cases,yielding an effective rate of 57.14%(20/35).The Gesell Developmental Schedule was employed to conduct a psychosocial assessment of the pediatric patients.Out of the total of 35 cases,11 cases(11/35,31.4%)were classified as having a normal development with a DQ(Developmental Quotient)>85 and as being in the marginal range,with a DQ ranging from 55 to 84..Furthermore,24 cases(68.6%)of the patients exhibited a DQ below 55,indicating the presence of developmental delays of varying degrees.Conclusion:1.Infantile epileptic spasms syndrome is associated with immune dysregulation.2.The use of adrenocorticotropic hormone(ACTH)in the treatment of IESS may potentially modulate lymphocyte proportions through the HMGB-1/TLR4 pathway,control inflammatory factors,and suppress innate immunity.3.The HMGB1-TLR4 axis is closely associated with the development,progression,and effectiveness of ACTH treatment for IESS and may serve as a potential biomarker for IESS diagnosis and treatment.