Idiopathic Thrombocytopenic Purpura

B-cell disorders include a large group of malignant and nonmalignant diseases with tremendous variation in incidence, natural history, treatment, and prognosis. This article will focus on adult idiopathic thrombocytopenic purpura (ITP) and chronic lymphocytic leukemia (CLL). Clinicians must individualize treatment for ITP and CLL to each patient. Observation without intervention is appropriate for some patients, whereas immediate treatment is indicated for others. Deciding when to treat and which agents to use can be diffi cult, but new therapeutic options are emerging for both conditions. Emerging Therapeutic Options for B-Cell Disorders: Idiopathic Thrombocytopenic Purpura and Chronic Lymphocytic Leukemia

Background: CD 4 + CD 25 + T cells play an important role in the induction and maintenance of peripheral self-tolerance. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion.
Objective: To investigate the role of CD 4 + CD 25 + regulatory T cells in children with idiopathic thrombocytopenic purpura (ITP) and correlate their levels with different disease stages, this study was conducted in 45 children (15 controls and 30 cases with ITP); 10 of these patients were in the acute phase (Group I), 10 in the remission phase (Group II), and the remaining 10 in the chronic phase (Group III).
Methods: Studied groups were subjected to the following investigations: complete blood count, erythrocyte sedimentation rate, Coombs test, and flow cytometric analysis of CD 4 + CD 25 + T lymphocyte count.
Results: There was a highly significant decrease in platelet count and CD 4 + CD 25 + count in patients of the acute group when compared with remission, chronic, and control groups. There was a significant decrease in CD 4 + CD 25 + count in the chronic group when compared with remission group. In addition, a significant decrease in CD 4 + CD 25 + count was found in remission group when compared with the control group. Moreover, there was a positive correlation between CD 4 + CD 25 + count and platelet count in all the groups; this correlation was significant in the acute group and insignificant in the remission and chronic groups.
Conclusions: Decreased number of CD 4 + CD 25 + T cells might be one of the mechanisms that cause immune regulation dysfunction in ITP and the count of CD 4 CD 25 T cells is considered to be related to the severity of ITP, as these cells were significantly decreased in the active phase of the disease and increased in the patients at the complete remission phase. Therefore, count of CD 4 + CD 25 + T cells might be a helpful diagnostic predictor of onset and improvement of ITP in children. Finally, it is suggested that these cells may have therapeutic implications that need further clarification. I diopathic thrombocytopenic purpura (ITP) is characterized by the production of autoreactive antibodies to platelet antigens, resulting in both accelerated destruction of platelets and reduced platelet production. Although healthy individuals harbor platelet-specific autoreactive T cells that are tolerized in the periphery, patients with ITP possess activated platelet autoreactive T cells and cytokine imbalance, suggesting loss of peripheral tolerance in ITP patients. CD 4 + regulatory T (T reg ) cells play an important role in the maintenance of peripheral tolerance and are characterized by the expression of the CD 25 surface marker and the transcription factor forkhead box protein 3 (Foxp3), making up 5% to10% of the normal CD 4 + T-cell population. 1 The most common antigenic epitopes for antiplatelet antibodies in ITP are the platelet GPIIb/IIIa and GPIb-IX receptors. By binding to these specific sites on the platelet surface, antibodies opsonize platelets for clearance by the cells of the reticuloendothelial system, primarily in the spleen. 2 Autoreactive T cells have been identified in patients with various systemic or organ-specific autoimmune diseases. 3 On the other hand, autoreactive T cells are also present in the peripheral blood of a healthy individual without any evidence of autoimmune disease. These findings suggest that some mechanisms exist, which regulate these autoreactive T cells to prevent autoimmune disease. 4 CD 4 + CD 25 + T cells play an important role in the induction and maintenance of peripheral self-tolerance. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. 5 The CD 4 + CD 25 + T reg cells are characterized by the constitutive expression of several activation markers, including CD 25 , cytotoxic T lymphocyte-associated antigen-4, glucocorticoid-induced tumor necrosis factor receptor family related gene, lymphocyte activation gene-3, and programmed death-1. These molecules are predominantly expressed at an elevated level on CD 4 CD 25 T reg cells, which allows for their discrimination and subsequent isolation for functional studies. 6 CD 4 + CD 25 + T reg cells have a significant role in orchestrating the development of immune responses and deficiency of T reg cells may lead to a great risk of peripheral tolerance breakdown and development of autoimmunity. 7

AIM OF THE STUDY
The aim of the present study was to evaluate the role of CD 4 + CD 25 + T reg cells in children with ITP and correlate their levels with different disease stages. pediatric department of Suzan Mubarak University Hospital for Children at El-Minia University. In addition, 15 apparently healthy children were enrolled as control group.
The cases included in our study were grouped as follows: Group I: GI (acute cases) comprised 10 patients, 4 males and 6 females, aged between 2 and 6 years. All patients in this group were diagnosed as acute ITP by sudden onset 8 and duration less than 6 months, typically from 1 to 2 months. 9 Group II: GII (remission cases) comprised 10 patients, 5 males and 5 females, aged between 3 and 9 years. All patients in this group had an acute ITP and were in the remission stage. Complete remission is defined as having a normal platelet count and disappearance of symptoms 10 for at least 6 months. Group III: GIII (chronic cases) comprised 10 patients, 3 males and 7 females, aged between 9 and 12 years. All patients in this group were diagnosed as chronic ITP by insidious onset 8 and duration of more than 6 months. 9 Group IV: GIV (control group) comprised 15 apparently healthy children, 9 males and 6 females, aged between 3 and 12 years.

Inclusion Criteria
Patients with idiopathic thrombocytopenia (platelet count <150,000) and purpura with no other proved cause of thrombocytopenia or pupura were included in the study.
All patients and control groups were subjected to: (A) Detailed history taking including past history of trauma or drugs. (B) Complete clinical examination with special stress on rashes, signs of bleeding, hepatosplenomegaly, and lymphadenopathy. (C) The following laboratory investigations were carried out: 1. Complete blood cell count (red blood cells, white blood cell, platelets) using an automated cell counter, Sysmex NE. 2. Erythrocyte sedimentation rate by Westergren method (for patients only). 3. Coombs test (for patients only). 4. Bone marrow aspiration (for patients only). 5. CD 4 + CD 25 + T cell counting using dual-color reagents are based on the combination of 2 antibodies labeled with fluorescein isothiocyanate and R-phycoerythrin. This combination is particularly effective as both the fluorochromes can be excited at 488 nm. The fluorescence emission for fluorescein isothiocyanate is in the green region (around 530 nm), whereas the R-phycoerythrin emission is in the orange region (above 570 nm) supplied by Dako Company on coulter flow cytometer. 11

Blood Sampling
Under complete aseptic conditions, 3 mL of venous blood was drawn from each patient and divided into: 1.6 mL of blood in a dry plastic tube [which was added to 0.4 mL of sodium citrate for erythrocyte sedimentation rate] and 1.4 mL of blood on EDTA tube [for complete blood count, Coombs test, and flow cytometric assessment of CD 4 CD 25 T cells].

Statistical Analysis
Data entry and analysis were done with IBMcompatible computer using statistical package of social science (SPSS) software for Windows version 13. Graphics were done using Excel. Quantitative data were presented by mean and standard deviation, whereas qualitative data were presented by frequency and distribution. Correlations, w 2 , Student t test of proportion, and 1-way analysis of variance were done. The probability of less than 0.05 was used as a cut off point for all significant tests.

RESULTS
There were no significant differences regarding age and sex among the studied groups. Also, comparison of WBC and lymphocyte counts among the 4 groups revealed no significant differences. Erythrocyte sedimentation rate (first and second hours) was within normal limits and Coombs test was negative in patients of all groups.
There was a highly significant decrease in the platelet count in the acute group when compared with remission, chronic, and control groups. Also, there was a highly significant decrease in platelet count in the remission group in comparison to the control group. In chronic patients, there was a highly significant decrease in platelet count compared with remission and control groups (Table 1).
There was a significant decrease in CD 4 + CD 25 hi + cells in patients of the acute group when compared with patients of the remission, chronic, and control groups. There was a significant decrease in CD 4 + CD 25 hi + cells in patients of the chronic group when compared with remission group. Moreover, there was a significant decrease in CD 4 + CD 25 hi + cells in remission group when compared with control group. Also, a significant decrease in CD 4 + CD 25 hi + cells was detected on comparing chronic group with control group (Table 2).
There was a significant decrease in CD 4 + CD 25 low + cells in patients of the acute group when compared with patients of the remission, chronic, and control groups. There was a significant decrease in CD 4 + CD 25 low + cells in patients of chronic group when compared with patients of remission group. Moreover, there was a significant decrease in CD 4 + CD 25 low + cells in patients of remission group when compared with patients of control group. Also, a significant decrease in CD 4 + CD 25 low + cells was observed on comparing chronic group patients with control group patients (Table 3). Regarding CD 4 /CD 25 ratio, nonsignificant differences between the studied groups were detected.

Correlations in Group I
A significant positive correlation was observed between platelet count and CD 4 + CD 25 hi + in the acute group (r=0.81, P=0.004. In addition, there was a significant positive correlation between platelet count and CD 4 + CD 25 low + % (r=0.4, P=0.01).

DISCUSSION
ITP was first described by Werlhof in 1735 as Morbus Maculosus Hemorrhagicus. 12 The pathophysiology of the disease is known to be immune-mediated. Direct T-cell mediated cytotoxicity against megakaryocytes and platelets is the primary mechanism of thrombocytopenia. 9 In contrast to the naturally occurring CD 4 + CD 25 + T reg cells, inducible CD 4 + T reg cells are also thought to develop from conventional CD 4 + CD 25 + T cells in the periphery during activation in vivo or in vitro. These include IL-10 producing type 1 T regulatory (Tr1) cells and transforming growth factor (TGF)-b-producingTh3 cells. CD 4 + CD 25 + T reg , Tr1, and Th3 cells may cooperate in vivo to prevent aberrant immune response. Of the 3 different populations, CD 4 + CD 25 + T reg cells are the best studied because these cells can be obtained ex vivo with ease. 13 15 In vivo studies by Nehls et al 16 on the role of T reg cells in autoimmune diseases have been conducted in different murine model systems. When genetically susceptible mice were thymectomized on the third day of their lives, they developed various organ-specific autoimmune diseases and on transferring CD 4 T cells from a normal adult mice to the thymectomized mice on the 14th day of life, the development of these diseases were inhibited.
The present study was conducted to further investigate and clarify the role of CD 4 CD 25 T reg cells in children with ITP during acute, remission and chronic stages.
In the present study, there was a significant decrease in platelet counts among all groups of ITP (acute, remission, and chronic) when compared with control group. Also, there was a significant decrease in platelet count in acute group when compared with remission and chronic groups. Concomitantly, there was a significant decrease in CD4% and CD25% in acute, remission, and chronic ITP groups when compared with the control group.
The results of the present study are supported by other investigators; Fahim and Monir 17 who investigated the functional role of CD 4 CD 25 T reg cells in childhood immune thrombocytopenic purpura. By using flow cytometry, they found that the number of CD 4 CD 25 T reg cells was significantly decreased in acute and chronic ITP patients as compared with controls, and they concluded that CD 4 CD 25 T reg cells are critical in maintaining self tolerance and preventing organ specific autoimmune diseases. Finally, they recommended that these T reg cells might form a base for specific immunomodulatory therapies for ITP in future.
In addition, Liu et al 18 explored the profile and function of CD 4 CD 25 T reg cells in ITP patients. The percentage of T reg cells was significantly decreased in ITP patients in active and nonremission phase (5.79% ±1.22%) when compared with the patients in remission phase (11.63%±4.56%) and with healthy individuals (12.68%±3.59%). These results suggested that the decreased number and function of T reg cells might be one of the mechanisms that cause immune regulation dysfunction in ITP.   The explanation of lower CD 4 CD 25 in ITP patients can be driven from the critical role of regulatory cells in self tolerance and autoimmunity, which has been clearly established in thymus-derived CD 4 CD 25 T reg cells. 20 Expansion of this explanation can be driven from the facts published by other investigators, which indicate that the immune system employs several strategies to maintain its own homeostatic balance. The thymus plays an important role in this regulation, because of its particular role in clonal deletion of self-reactive T cells, but other mechanisms preventing immunological self attacks are also involved in the maintenance of self tolerance. These latter mechanisms occur in the periphery and include T-cell anergy, T-cell ignorance, and the effects of regulatory cells. T reg cells can be divided into 2 groups, those that are anergic and require cell-cell contact for their function, and those, including Th 1 and Th 3 cells, functioning by the secretion of inhibitory cytokines. 21 Immunoregulatory CD 4 CD 25 T cells play an important role in the induction and maintenance of peripheral self-tolerance. These professional regulatory cells prevent the activation and proliferation of potentially autoreactive T cells that have escaped thymic deletion. Therefore, CD 4 CD 25 T cells (T reg cells) are believed to possibly play an important role in pathogenic autoimmune diseases. 5 Several studies suggested that circulating T reg cell frequency and/or function may be used as a marker for evaluating autoimmune status in patients. 22 Other studies in patients with ITP have shown reduced levels of Foxp3 mRNA 5,18 and protein in circulating mononuclear cells and abnormal T reg function in spleen biopsies. 18 A study was conducted by Yu et al, 23 which investigated the frequency and function of circulating CD 4 + CD 25 + hi T reg cells in patients with chronic ITP and revealed a comparable frequency of circulating CD 4 + CD 25 + hi FoxP3 T reg cells in patients and controls. However, sorted CD 4 + CD 25 + hi T reg cells from patients with chronic ITP had a 2-fold reduction of in vitro immunosuppressive activity compared with controls.
These studies indicate that deficiency in generation and/or defective functions of T reg cells may contribute to loss of immunologic self-tolerance in patients with ITP. However, we did not assess Foxp3 in our study. The ability to isolate and expand T reg cell populations with immune suppressive activity will enable new forms of adoptive immunotherapy with a wide variety of clinical applications. It is likely that suppressor T cells will first be tested for their ability to prevent or treat graft-versus-host disease after allogenic bone marrow or stem cell transplantation. A more technically challenging approach will be the use of T reg cell therapy for autoimmune disorders. 24

CONCLUSIONS
The results of the study suggest that decreased number of CD 4 CD 25 T cells might be one of the mechanisms that cause immune regulation dysfunction in ITP. Furthermore, the results confirm that CD 4 CD 25 T-cell count is related to the severity of ITP, as these cells decreased significantly during the active phase of the disease whereas they increased during the complete remission phase. Therefore, CD 4 CD 25 T-cell count might be a helpful diagnostic predictor of onset and improvement of ITP in children. Finally, we suggest that these cells may have therapeutic implications that need further clarification. Further studies are recommended on a larger scale of ITP patients treated with different regimens to determine the effect of these lines on enhancement of the number and activity of CD 4 CD 25 T reg cells and to evaluate the use of CD 4 CD 25 T reg cells as a potential therapeutic tool in the treatment of autoimmune diseases.