الفهرس 
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Abstract
Type 1 diabetes (T1DM) is a chronic auto immune disease resulting in destruction of pancreatic β cells and absolute insulin deficiency with subsequent hypoinsulinemia and hyperglycemia. It is most often diagnosed in children and adolescents. The incidence of T1D increases with age with a peak around the age of 10 years but with no sex predominance. In Mediterranean and Middle East countries, the highest incidence is in Egypt. T1DM is believed to be caused by a complex interplay between genetic predisposition, environmental factors and the immune system.
An imbalance between Teffector cells (Teff) and T regulatory cells (Treg) is an identified cause for autoimmunity. In T1DM, NK cells initiate pancreatic islets cell lyses. Loss of Tregs at disease onset facilitates activation and accumulation of NKs in the pancreatic microenvironment. The natural cytotoxicity receptor (NCR1) is found to be expressed on all NK cells and represents a very accurate measure of NK. A proper low dose IL-2 could enhance Tregs and enforce control and regulation of proinflammatory NKs. The role played by NK cells needs to be extensively studied in order to improve therapeutic strategies intended at resetting the balanece between Teff and Tregs.
The present work employed novel formulations of low dose IL-2 loaded on chitosan nanoparticles. The study included 115 male inbreed BALB/c mice induced for diabetes by multiple low dose Streptozotocin injections. Following induction of Diabetes, mice were injected with different doses of IL-2 and control formulations (empty chitosan). Diabetic satatus were monitored by measuring mice weights, plasma glucose and serum insulin. Splenocytes were maintained in a short-term culture for in vitro assessment of spontaneous versus Con A induced production of NCR1 and FOXP3 levels as relevant markers of NK and Treg cells activity; respectively.
The present work showed the following results:
Plasma blood glucose levels were checked once before and three weeks after STZ injection and IL-2 treatment. Insulin levels were measured with animal weights to assess diabetic status of mice.
Concerning weight variations, the statistical analysis of the results pointed out to the presence of signifant differences due to STZ injection as compared to controls. STZ-treated mice showed notable weight loss at weeks 1 and 2 after IL-2 treatment which wasn’t apparent at week 3. The recovery was accelerated in the presence of IL-2 treatment (either free or chitosan loaded) in all doses. Fasting serum glucose and insulin levels were compared to negative control mice. There was a significant decrease in plasma glucose in diabetic mice at week 1. The effect of IL-2 treatment was random in some groups that showed relatively higher glucose levels irrespective of being treated with free or chitosan loaded IL-2. At week 2, glucose level was elevated in the diabetic group and treatment with free IL-2 was effective although
chitosan encapsulation of IL-2 wasn’t efficient at all in recovering glucose status. At week 3, the situation was improved where glucose level in mice treated with IL-2/chitosan wasn’t worsened in comparison to mice treated with free IL-2. Regarding insulin level in week 1, there was a significant improvement in diabetic mice treated with all concentrations of free IL-2 and also in diabetic un-treated mice. IL-2 encapsulation into chitosan was not effective where insulin deficiency was worsend. At weeks 2 and 3, the improvement in insulin level by IL-2 wasn’t significant.
In the present study, Fox-p3 was assessed in culture supernatants harvested after a short term culture of splenocytes either with or without mitogenic stimulation by Con A. There was no significant changes observed in Fox-p3 in diabetic un-treated groups as compared to negative controls in any of the three week intervals either at basal or mitogen-induced activity. At week 1, there was a significant increase in basal Fox-p3 due to IL-2/chitosan treatment particularly at the dose of 0.05 rather than 0.1 and 0.3. Significant change in mitogen-induced Fox-p3 on the other hand was restricted to diabetic mice treated with free IL-2 at dose of 0.3 that showed higher Fox-p3 expression when compared to mice treated with the same doses of IL-2 loaded onto chitosan. Week 2 variations in basal Fox-p3 were concentrated in diabetic mice treated with IL-2/chitosan at a dose of 0.3 where a significant decrease was recorded in comparison to diabetic un-treated and IL-2/Chitosan regardless of the dose. Following mitogen stimulation, chitosan loaded IL-2 treated diabetic mice showed a significant reduction in Fox-p3 as compared to those treated with empty chitosan. Week 3 of the study revealed a significant increase in basal Fox-p3 in mice treated with IL-2/chitosan at dose of 0.3 as compared to mice treated with empty chitosan.
In the current study, NCR1 was assessed in culture supernatants gathered after a short term culture of splenocytes before and after mitogenic stimulation by Con A. Results of 1st week indicated that treatment of diabetic mice with recombinant IL-2 loaded onto chitosan at a dose of 0.3 nanoparticles induced a significant spontaneous improvement in NK cell activity in comparison to negative control mice, diabetic mice as well free IL-2 0.3, IL-2/chitosan at doses of 0.05 and 0.1. After mitogenic stimulation there was a significant increase in IL-2/chitosan at a dose of 0.1 than diabetic untreated mice and IL-2/chitosan 0.05. Variations in week 2 was restricted to basal NCR1 activity in mice treated with IL-2/chitosan at dose of 0.1 when compared to negative controls, diabetic mice and IL-2/chitosan at a dose of 0.05. Mitogen stimulation of the same week revealed statistical decrease in IL-2/chitosan 0.05 when compared to higher concentrations (0.1 and 0.3). Analysis of results at week 3 either basal or after mitogenic stimulation didn’t show any significant differences in NCR1.
Correlation studies undertaken among different study parameters of the currentstudy revealed the following:
1-A significant positive correlation between insulin level and weight at the 1st week.
2- A significant negative correlation between insulin level and basal level of NCR-1 and a significant positive correlation was also rercorded between basal level of Fox-p3 and both weight and basal level of NCR-1 at the 1st week.
3- A significant negative correlation between mitogen-stimulated NCR-1 results and both insulin and weight and also a significant positive correlation between mitogen-stimulated Fox-p3 and weight at the 1st week.
4- A significant positive correlation between insulin level and weight at the 2nd week.
5-A significant negative correlation between weight and basal NCR-1 levels and asignificant positive correlation was also rercorded between basal Fox-p3 results and insulin level while it tested a significant negative correlation with weight at 2nd week.
6-A significant negative correlation between mitogen-stimulated NCR-1 and glucose at 2nd week.
7-A significant negative correlation between insulin level and weight and a significant positive correlation between insulin level and glucose at 3rd week.
8-A significant negative correlation was rercorded between glucose and basal NCR-1 levels anda significant negative correlation was also rercorded between basal Fox-p3 and NCR-1 at 3rd week.
9-A significant negative correlation was recorded between mitogen-stimulated NCR-1 and both insulin and glucoseat 3rd week.
10- A significant positive correlation between FOX-P3 and NCR-1 during 1st week specifically inIL-2 loaded chitosan with lowest dose (0.05) and free IL-2 in the highest dose (0.3).
Conclusions:
1- Intraperitoneal injection of streptozotocin caused a rapid and brief hyopoglycemia followed by state of transient and mild hyperglycemia which was associated with hyperinsulinemia and weight loss.
2- The induced hyperglycemia was remarkable at week 3 following IL-2 treatment.
3- The results of glucose and insulin weren’t homogenous indicating that the mechanism of diabetes induction following STZ was not insulin-dependent.
4- It was also observed that Fox-p3 didn’t show any significant variation indicating that diabetes induction not attributed only to controlled T reg cell activity but may also be mediated by other cells.
5- Following treatment with IL-2, it was clearly noted that curative effect of IL-2 was restricted to free form of IL-2.
6- Concerning the NK cell marker (NCR1), the results were more consistent where chitosan encapsulation of IL-2 showed enhancing effect on NCR1 expression relative to the free IL-2 at doses of 0.1 and 0.3.
7- Prolonged administration of low-doses IL-2 results in enhanced expression of NCR1 indicating a significant role of Tregs in NK activation and function by controlling the availability of IL-2 in the microenvironment.
8- Low-dose IL-2 selectively modulates NCR1 NK and FOXP3+Tregs and increases their expressions.
Recommendations
1. Modification of the adopted protocol of T1DM induction by STZ, in terms of drug dose, number of injections, duration and strain of the experimental animals might be of interest in standardizing the conditions required for optimal induction of the disease.
2. Conducting the study on higher number of mice to improve the statistical power for analysing mechanistic and biomarker data.
3. Study the expression of NCR1 and FOXP3 surface expression on NK and Tregs cell populations rather than at the cell lysate level would be of more meaningful significance.
4. An important concern remains the possible interference of eosinophil activation induced by low doses of IL-2 therapy. This may pave the way for optimization of IL-2 therapy protocols in T1D aiming at enhancing antigen-specific Tregs.
5. Standardizing the contribution of IL-2 therapy on a wide range of other autoimmune disorders would be important in foundation of a future of precision medicine.
6. Future studies directed towards comprehensively mapping the IL-2 signaling network and further elucidating the molecular mechanisms of IL-2 action to facilitate the development of specific agonists and antagonists and efficiently modulate immune responses.
7. Conducting functional assays to indicate a role for Tregs in NK homeostasis, activation and function by controlling the availablility of IL-2 in microenvironment.