الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
The present study was performed to establish a correlation between the
alteration in the signaling pathways of type I Interferon and the perturbation in B and T
lymphocyte proliferation, existence and functions during type I diabetes mellitus.
Firstly, Experimental type I diabetes mellitus is produced in group by the administration
of Streptozotocin (60 mg/kg), which damages the insulin-producing islet cells of the
pancreas
1. Total numbers of 60 adult male group weighting 20-30 g were used in this
experiment. Group maintained under standard laboratory conditions, fed a diet of
standard commercial pellets and given water and libitum. All group were fasted
for 20 hr before diabetes induction. They divided equally into three groups: The
first group served as a control (n = 20) were injected with the vehicle alone (0.01
M citrate buffer, pH 4.5), the second group (n = 20) were rendered diabetic with
an intraperitoneal injection of a single dose of STZ (60 mg/kg body weight) in
0.01 M citrate buffer (pH 4.5), while the third group (n = 20) were rendered
diabetic treated group with an intraperitoneal injection of a single dose of STZ (60
mg/kg body weight) in 0.01 M citrate buffer (pH 4.5) and then injected with an
intraperitoneal anti-IFNAR1 solved in distilled water subcutaneously in a dose of
10 mg/kg body weight once/day for up to 20 days according to National Institute
of Health (NIH) protocol.
2. To optimize all the parameters and conditions of the group models during the
experiments, blood glucose levels were monitored in all groups of group
throughout the experimental period. The diabetic group exhibited an obvious and
significant increase in the levels of glucose to control non diabetic group.
Moreover, it was observed that blocking type I IFN receptor in diabetic group
partially and significantly restored the altered levels of blood glucose as
compared to diabetic non-treated group.
3. Confirmed to the previous results, histological study of pancreas from STZinduced
diabetic group demonstrated atrophic islets of Langerhans as well as a
significant disturbance in the architecture of pancreatic langerhans cells as
compared to control and diabetic treated group. Furthermore,
immunohistochemical study using anti-insulin antibody for demonstration of Beta
cells in the pancreatic langerhans of the STZ-induced diabetic group showed a
significant highly reduction in the numbers of β cells which are responsible for
the production of insulin as compared to control non diabetic group. Interestingly,
blocking type I IFN receptor in diabetic group partially restored numbers of β
cells as compared with diabetic non-treated group was observed.
Secondly, In the present study, observed significant increase in the level of IFN in the
blood of diabetic group as compared to control. While blocking type I IFN receptor in
diabetic group partially and significantly restored the altered levels of IFN-α as
compared to diabetic non-treated group. Moreover, proinflammatory cytokines as IL-1α,
IL-1β, IL-6, TNF-α and CXCL10 (IP-10) levels were determined in an ELISA assay for
every group. It was observed that aberrant and significantly elevated level of IL-1α, IL-
1β, TNF-α and IL-6 in diabetic group compared with the control which indicated a
prolonged pro-inflammatory stimulus during diabetes. Furthermore, blocking type I IFN
signaling during diabetes significantly and partially restored the levels IL-1α, IL-1β, IL-6,
TNF-α and CXCL10 (IP-10) during diabetes indicated that interferon alpha induced the
production of proinflammatory cytokines during diabetes. Thus, the present study
revealed that IFN-α activates the peripheral immune system and induces the production
of proinflammatory cytokine during type I diabetes mellitus.
Thirdly, to further explore affects of IFN-α on lymphocytes proliferation during type I
diabetes mellitus.
Isolated PBMCs from three groups were harvested and washed twice in PBS
and then stained with 0.63 mM carboxyfluorescein diacetate succinimidyl ester (CFSE)
for 8 min at room temperature, CFSE-labeled cells were seeded in 6-well plates and
treated with or without cocktail mitogen and grown for 4 days in cell culture medium.
The CFSE fluorescence intensity was measured by flow cytometry analysis. This study
investigated whether the ability of PBMCs to proliferate in response to mitogen. It was
observed that the proliferative capacity of PBMCs was significantly decreased in
diabetic group as compared with the control group and anti-IFN-treated diabetic group.
Moreover, to confirm the previous results, the cell cycle of each group was analyzed
using propidium iodide single staining method and flow cytometry analysis. The
percentage of cells undergoing apoptosis was determined using flow cytometry as the
percentage of hypo diploid cells (sub G0/G1 peak). Dead cells were determined with the
trypan blue exclusion test. It was found that the percentage of apoptotic cells was
significantly increased in diabetic group compared with control. Blocking type I IFN
significantly reduced the percentage of apoptotic cells.
Fourthly, in order to explore the signaling pathways of IFN-α downstream IFNAR
involved in the apoptotic effects of IFN-α on lymphocytes during type I diabetes mellitus.
1. PBMC isolated from 5 groups from each group were stimulated with IFN-α (2000
IU/ml) for 5 minutes or without stimulation and cell lysates were prepared for
western blot analysis. The cell lysates were incubated with 0.001% of primary
antibody of the phospho-STAT1, STAT1, phosphor-STAT2, STAT2, phospho-
IκB, IκB ,AKT and phospho-AKT (4 ml of blocking buffer (4%) + 4 μl of primary
antibody solution derived from rabbit) overnight on a rocker or orbital shaker at
4oC.
2. It was found that the PBMC isolated from diabetic group were continuously, In
vivo, stimulated with high level of type I IFN. Moreover, stimulation of PBMC
isolated from all groups of group with IFN-α revealed significant phosphorylation
of AKT, IκB-α, STAT1 and STAT2. Nevertheless, if subtracted the normalized
phosphorylation of these proteins without stimulation from their values upon IFNstimulation,
this revealed that type I IFN signaling during diabetes is clearly
perturbed observed as aberrant and continuous phosphorylation of these
transcraption factors. Interestingly, diabetic group that were treated with anti-
IFNAR1 exhibited normal signaling as compared with control non diabetic group.
Moreover, the role of IFN-α in the continuous activation and exhaustion of
lymphocytes during type I diabetes mellitus was abolished by PD-1 expression using
flow cytometry analysis. Therefore, it was observed up-regulation of PD-1 expression on
the PBMCs that were isolated from diabetic group revealed that these cells are
exhausted. Blocking type I IFN clearly restored the functional state of these cells.
Fifthly, in order to investigate the effect of blocking type I IFN receptor signaling
pathway during TID on the lymphocyte proliferation and functions within lymphoid
organ. Histological and immunohistochemical studies for spleen and peyer’s patches as
a secondary lymphoid organs, while electron microscope for thymus, spleen and
peyer’s patches were done.
Light microscope data showed enlarged high-endothelial venules that clearly
observed in the peyer’s patches of the diabetic group as well as Immunohistochemical
study of diabetic group demonstrated reduced number of CD20+ B cells, CD3+ T cells,
CD8+ T cells and MHC class II in the spleen in comparison to control. In contrast, there
was an increase in CD3+ T cells, CD8+ T cells and MHC class II in the peyer’s patches
in comparison to that in control. Moreover, blocking type I IFN receptor restored number
of these cells more or less similar to the control. Furthermore, Immunohistochemical
study for PCNA demonstrated reduced number of immune cells in the proliferation
status within the spleen and peyer’s of diabetic group as compared to control group.
Thus, blocking type I IFN receptor restored number of these cells more or less similar to.