Role of leptin in immune thrombocytopenic purpura in children

Introduction
ITP is characterized by isolated thrombo-cytopenia, which refers to a platelet count below 100,000/µl, while maintaining normal levels of white blood cells and hemoglobin. The cause of ITP is still unclear in the majority of patients, but it can be initiated by several environmental variables such as viral infection and immunologic triggers [1].

Immune thrombocytopenic purpura (ITP) is characterized by symptoms and indica-tors of low platelet count, including bleeding gums, nosebleeds (epistaxis), easy bruising, purpura, and bleeding in the brain (intracranial hemorrhage)[2].

The pathophysiology of ITP is highly intricate and varied. Recent developments in ITP research indicate that the pathophysiology of the disease involves an intricate disruption of the immune system. Multiple studies have demonstrated that anomalies in T lymphocytes, natural killer cells, dendritic cells, cytokines, program-mmed cell death, as well as oxidative stress, infection, and medications, are significant factors in the development of ITP [1]. 

Leptin, an extensive protein consisting of 167 amino acids, was initially characterized as a hormone-like cytokine. Adipose tissue 
synthesizes it and it binds to a receptor belonging to the class I cytokine receptor family[3]. 

Studies have demonstrated that leptin has an impact on both the innate and adaptive immune responses. It serves as a chemical signal that attracts neutrophils, eosinophils, and basophils. Leptin signaling triggers phagocytosis and stimulates the generation of both pro- and anti-inflammatory cytokines by monocytes and macrophages. Leptin has the ability to activate basophils to release type 2 cytokines IL-4 and IL-13, but only in specific circumstances. [4]

An increased concentration of serum leptin has been linked to acute infections and autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and immune thrombocytopenic purpura (ITP). Therefore, it has been previously hypothesized that leptin plays a significant role in the development of ITP and could be a potential target for treatment. [3] . 

Aim of the work
The aim of this study was to assess serum level of leptin in immune thrombo-cytopenic purpura among children and its clinical significance.

Subjects and methods
The study was conducted on eighty-eight cases: 40 apparently healthy individuals (group II) with matched age and sex who served as a control group. Also 48 ITP patients whom   diagnosed  according to International Working Group (IWG), they  were further  subdivided into 2 subgroups: group Ia (25 ITP children in  acute state who newly diagnosed within three months) and group Ib ( 23 ITP children  in chronic state who Lasting for more than 12 months   ). Patients were selected from pediatric hematological outpatient clinic at Minia University hospital. During the period from September 2022 to September 2023. The hospital ethics committee approved this study and a written consent was obtained from each patient (Approval number: 637 /2023, Date of approval: 20 February 2023). The patients are usually treated using Solubrid and IVIG drugs. Patients with other causes of thrombo-cytopenia and patients with diabetes mellitus were excluded from the study. Both patients and control groups were subjected to complete history taking, complete clinical examination and labor-atory investigations.  

Blood sampling protocol: about 8 ml of venous blood was withdrawn from each participant by using a disposable plastic syringe after disinfection of skin with isopropyl alcohol (70%) swaps, and this sample was divided as follows: (a) 1.8 ml of blood on a tube containing 0.2 ml trisodium citrate for detection of INR (dilution 9:1). (b) 1 ml in EDTA-containing tube for CBC.  Then 5 ml of blood was transferred into two plain tubes, each tube was allowed to be clotted for 2 hrs at room temperature and then Centrifuged at 1000xg for 20 min, the expressed serum of first tube was used for determination of renal function tests, liver function tests and viral markers. the remaining serum of the other tube was stored refrigerated at -20°C for assay of leptin.

Methods
CBC was determined by (SYSMEX XN-1000, TAO Medical Incorporation, Japan), prothrombin time, concentration and INR (PT, PC and INR) determined by  (Stago Compact CT Coagulation analyzer), renal function tests (urea and creatinine), liver function tests (ALT, AST, Albumin, total bilirubin and direct bilirubin) and random blood glucose determined by (Selectra PROXL16.8361 ELTECH GROUP Clinical Systems, GERMANY) and Viral markers (HCV antibody, HBsAg and HIV) determined by (Cobas e411 Hitachi High -Technologies Corporation, Japan). Serum leptin was assessed by enzyme–linked immunosorbent assay (EIA). The kit was supplied by   Bioassay Technology Laboratory (catalog no. DL-LEPHU), China. The analysis of the data was carried out using the IBM SPSS version 25 statistical package software (IBM; Armonk, New York, USA). 

Data was tabulated and presented using various tests: frequency, calculation, and the mean, standard deviation, Chi-square test for qualitative data between control and study (acute and chronic variables).  Spearman`s correlation was done for specific markers with others age, routine investigations, and lines of treatment in the control and study variables. A p value is less than 0.05 was considered significant. T-test for quantitative data between control and study variables and also   ANOVA test was used to compare between control and study (acute and chronic variables).  
Results
The age range in the patient's group was from 1 to 15 years with mean 8.96±3.75, In control group was from 1 to 15 with mean of 9.80±2.20. All studied groups showed that disease was more in females with the female percentage being (52%, 55%) in group I and group II respectively. There was no statistically significant difference in age and sex between all groups (as shown in table I).      
 

Scatterplot illustrates a negative correlation (means increase serum leptin led to decrease PLT): hypothetical data for the relationship between serum Leptin level and PLT related to group Ia and group Ib patients

Discussion
ITP is a type of autoimmune condition that is acquired, meaning it is not present from birth. It is defined by a low platelet count, specifically less than 100,000 platelets per microliter of blood. This low platelet count is caused by an imbalance between immune cells that are successful at clearing platelets and immune cells that regulate this process. As a result, platelets are cleared from the blood at a higher rate and the production of new platelets is impaired. [5] 

Leptin was initially identified as a hormone-like cytokine and has been demonstrated to impact both innate and adaptive immunity. The serum leptin level was elevated during the acute onset of ITP due to the interaction between leptin and the immune system. Leptin concentration increases during infection and inflammation. Proinflammatory cytokines elevate the levels of leptin in the bloodstream during an acute infection. In turn, leptin stimulates the production of cytokines from monocytes and macrophages. [6]

The present study showed that most of ITP cases was females in contrast to  Liu & Liu. [7] who found that most of cases was males this result may be attributed to the small sample size, short period of observation and follow up. 

Also we found that the  majority of studied patients had ecchymotic patches on the same line with AbdalJabbar et al.,[8] who illustrated that the most of studied patients had ecchmotic patches, however Liu & Liu et al.,[7] who noticed the purpura was the most common complaint .

Most of acute patients received pulse steroid in a hormony with  Liu et al.,[9] who found that the majority of acute patients received pulse steroid  high dose dexamethasone as a first line of treatment, however majority of chronic patients received  solumedrol in agreement with  Aydn & Gürkan. [10] who documented  that the majority of chronic patient take corticosteroid  due to low cost and convenience of continuous use, this result related to the purpose of use of corticosteroid  in ITP is to reduce antibody production by immunosuppression and prevent platelet destruction by macrophage / monocytes., also 39.1% of patients in chronic group received IVIG  in contrast to Ozcelik et al.,[11]  who  documented that low percent of chronic  ITP patients treated by IVIG due to life threatening side effect as renal failure and thrombosis. 

Our study found that platelets count was lower in acute group than chronic group and control group  on  the same line with Hassan et al., [1] who  documented that platelets count was lower in  acute than chronic ITP patients furthermore, Karaman & Doğan.[12] who observed that platelet count at diagnosis is an important laboratory predictor of ITP course. And there were significantly lower platelet counts among the patients with  acute onset of ITP and chronic ITP.

As shown in this work  serum leptin level was higher in acute and chronic ITP than  control group  on the same line with Xu et al.,[13]   who noticed significantly higher serum leptin in  rapid onset of acute ITP patients  and  chronic  child patients  than control  group  which involved in patho-genesis in ITP, also  serum leptin was higher in acute than chronic ITP patients in agreement with Thomas et al.,[3] who noticed that serum leptin increase in acute ITP  patients compared with chronic group which involved in the pathogenesis and diagnosis of childhood ITP patients. Furthermore, leptin has a direct effect on immune function. [6]

The results of our study support the notion that leptin has an anti-inflammatory role in children with ITP. This is because high levels of leptin in the blood serve as a soluble mediator during the acute phase of this autoimmune disease. [3]

Frasca et al.,[14] found that leptin plays a proinflammatory function in the develop-ment of autoimmune illnesses. This is because leptin in the blood interacts with the immune system and its levels rise during infection and inflammation. During the onset of inflammation, the levels of pro-inflammatory cytokines rise, leading to an increase in the amount of leptin in the bloodstream. Leptin, in turn, enhances the release of cytokines from monocytes and macrophages. The role of leptin in modulating immunological function in humans is highly supported by the higher occurrence of ITP pathogenesis. The results of our study demonstrated a statistically significant negative correlation between platelets and serum leptin in both the acute and chronic groups, which aligns with the findings of Thomas et al.,[3]. They also observed a statistically significant negative correlation between platelets and serum leptin in patients with acute and chronic conditions due to a positive correlation between serum leptin and platelet-associated antibodies (PA IgG and PA IgM) during the acute phase of ITP.  

Furthermore, a statistically significant association was observed between serum leptin and IVIG in the patient group, which is in contrast to the findings of Thomas et al.,[3], who reported no link between serum leptin and therapy with IVIG in acute ITP.  
There was a statistically significant link between serum leptin and solumedrol in the acute group, as indicated by Thomas et al.,[3]. They reported a favorable correlation between corticosteroid treatment and serum leptin in acute ITP.