الفهرس 
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Abstract
Type 1 diabetes mellitus is caused by deficiency of insulin secretion due to pancreatic β-cell damage. It is the most common endocrine-metabolic disorder of childhood and adolescence, with important consequences for physical and emotional development. Diabetes complications represent a huge burden for patient and health services. The fight against each single complication has led to significant improvement in diabetes care, especially for micro-vascular complications, yet, they remain a major source of morbidity and mortality. A common approach for the prevention and treatment of diabetes complications relies on the understanding of their complex pathophysiology.
The pathophysiology of this disease involves failure in immune regulation. Human T regulatory cells (Tregs) are CD4+CD25high T cells and play an important role in controlling autoimmunity. Their qualitative or quantitative dysfunctions may result in ineffective suppression of autoreactive T cells that are responsible for the gradual destruction of insulin-producing cells. Low percentage of Tregs with CD62L (L-selectin) expression and their poor efficacy in preventing autoimmunity was observed in non obese diabetic mouse. Another surface molecule which seems to be important for regulatory T cells is the tumor necrosis factor receptor type 2 (TNFR2). Chronic inflammation present in autoimmune environment may impair the number and/or function of Tregs, which then are not able to prevent the activity of inflammatory cells. As inflammation proceeds, the risk for vascular complications increases. Tregs expressing CD62L or TNFR2 have been reported to be associated with inflammatory conditions that are present in patients with type 1 diabetes.
In view of these data, this study aimed was to determine the percentage of peripheral blood regulatory T cells expressing CD62L and the percentage of regulatory T cells expressing TNFR2 in patients with type 1 diabetes mellitus and assess their relation to the clinicopathological characteristics of patients and micro-vascular complications correlating their levels with markers of inflammation and metabolic control.
This cross sectional study was carried out on 50 children and adolescents with type 1 diabetes mellitus (14 males and 36 females with a male-to-female ratio of 1:2.6) attending the Pediatric Diabetes Clinic, Pediatric Hospital, Ain Shams University. Another group of 30 age- and sex-matched healthy individuals; 10 males and 20 females (ratio: 1:2) with no obvious medical disorders and not receiving any medication were enrolled as a control group. The mean age of patients was 14 ± 2.7 years (range: 9-19 years) while that of controls was 13.4 ± 1.9 years (range: 9-17.5 years).
Patients were excluded if having any clinical evidence of infection (CRP >10 mg/L), history of allergies, rheumatoid arthritis, recent trauma, surgery, physical inactivity, hematological diseases, liver dysfunction, urinary tract disorders, connective tissue disease, or other autoimmune disorders and any other conditions that could influence CRP. Patients taking antihypertensive drugs (rather than Captopril for patients with nephropathy), anti-platelet or lipid lowering medications at the time of the study were also excluded.
All the included patients were subjected to: (i) detailed medical history with special emphasis on age of onset of diabetes, disease duration, insulin therapy, acute metabolic complications; symptomatic hypoglycemic attacks or episodes of diabetic ketoacidosis and chronic diabetic complications (retinopathy, neuropathy, nephropathy, or cardiovascular ischemic events).; (ii) thorough clinical examination laying stress on anthropometric measures, blood pressure, fundus examination and neurological assessment; (iii) Laboratory investigations including measurement of mean random blood glucose (RBG), routine liver and Kidney function tests, fasting lipid profile, mean HbA1c%, urinary albumin excretion and high sensitivity C-reactive protein (hs-CRP). Determination of the percentage of regulatory T cells (Tregs), Tregs expressing CD62L and Tregs carrying TNFR2 using flow cytometry.
In the current work, it was observed that 23 patients out of 50 (46%) had complications; 7 (14%) patients had retinopathy, 9 (18%) patients had peripheral neuropathy and 15 (30 %) patients had microalbuminuria which was the most common chronic complication encountered in the studied patients.
Upon comparison of demographic data between all patients and control subjects, it was found that type 1 diabetic patients had significantly higher RBG, HbA1c, triglycerides, total cholesterol, urinary albumin excretion and hs-CRP with significantly lower HDL than control subjects (p<0.001). In addition, the percentage of regulatory T cells (CD4+CD25high T cells) and CD4+CD25high CD62Lhigh cells was significantly decreased while CD4+CD25highTNFR2+ T cells were elevated in diabetic patients compared with healthy controls (p<0.001).
Comparison between non-complicated and complicated type 1 diabetic patients and control group showed significant differences as regards age, height, systolic blood pressure, RBG, HbA1c, triglycerides, total cholesterol, HDL, urinary albumin excretion, hs-CRP and the percentage of CD4+CD25high cells, CD4+CD25high CD62Lhigh cells and CD4+CD25highTNFR2+ cells. Complicated diabetic patients had higher age, height, systolic blood pressure, RBG, HbA1c, triglycerides, total cholesterol, urinary albumin excretion and hs-CR than non-complicated patients and controls (p<0.05). Disease duration was longer and insulin dose was significantly higher in complicated patients compared with those with non-complicated ones (p<0.001). Non-complicated patients had significantly higher levels of all the studied laboratory variables compared with their healthy counterparts except for HDL cholesterol which was similarly distributed between groups.
The percentage of CD4+CD25high cells and CD4+CD25high CD62Lhigh cells was significantly decreased with increased frequency of Tregs expressing TNFR2 in patients with or without micro-vascular complications than control group (P<0.001). Similar results were obtained when the percentage of these cells were compared between complicated and non-complicated patients (p<0.001). ROC curve analysis revealed that the cutoff value of Tregs at 7.46%, Tregs expressing CD62L at 24.2% and Tregs expressing TNFR2 at 91.9% could differentiate complicated from non-complicated cases with a high sensitivity and specificity.
Analysis of regulatory T cells in relation to the clinico-pathological characteristics revealed a significant decrease in the percentage of cells in diabetic patients with micro-vascular complications and also, those with microalbuminuria (p<0.001) while the percentage of CD4+CD25high cells showed no significant difference in relation to age, sex and the presence of retinopathy or peripheral neuropathy (p>0.05). Analysis of CD4+CD25high CD62Lhigh T cells revealed similar results (P<0.001). On the other hand, Tregs expressing TNFR2+ showed significantly higher percentage among those patients (P<0.001).
Correlation studies revealed significant negative correlations between CD4+CD25high T cells and age, disease duration, insulin dose, RBG, HbA1c, urinary albumin excretion and hs-CRP. Similar correlations as that in CD4+CD25high T cells were obtained with respect to CD4+CD25high CD62Lhigh T cells (p<0.05) but age and RBG showed no significant correlations (p>0.05). On the other hand, significant positive correlations were found between Tregs expressing TNFR2 and disease duration, insulin dose, HbA1c, urinary albumin excretion and hs-CRP (p<0.05).
Multiregression linear analysis showed that HbA1c, urinary albumin excretion, and hs-CRP were independently related to the percentage of CD4+CD25high T cells in type 1 diabetic patients (r2 =0.9, p<0.001) while hs-CRP only was independently related to Tregs with CD62L or TNFR2 expression (r2 =0.701 and r2 =0.705, respectively; p<0.001).