الفهرس 
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Abstract
B-chronic lymphocytic leukemia (B-CLL) is one of the lymphoproliferative disorders which is characterized by accumulation of small-mature appearing lymphocytes in various compartments such as peripheral blood, bone marrow, lymph nodes and spleen. It was suggested that the monoclonal population of B-cells accumulates due to a reduction in the rate of apoptosis rather than to an increased proliferation rate.
It was shown that T-cell derived cytokines are very essential for regulating in vivo as well as in vitro survival and growth of the leukemic B cells. IL-4 is one of these cytokines which is involved in the survival mechanisms of these cells. It was shown that IL-4 protects B-CLL cells from spontaneous or drug induced apoptosis. Moreover, it appears that IL-4 production in vivo is the net result of T and B cells interactions as well as interactions among such cells and surrounding microenvironment.
B-CLL is still considered to be an incurable disease. Purine analogs are the first class of drugs capable of inducing apoptosis and may contribute to reduce the expression of proteins that are important for survival of B-CLL cells. Fludarabine the current standard treatment for B-CLL is one of these purine analogs, which induces apoptosis in normal and malignant lymphocytes. However, the exact mechanism underlying the apoptotic effect of Fludarabine is still a matter of great discussion.
So, the aim of the current work was to study the ability of Fludarabine to modulate IL– 4 level and its relevance to the apoptotic status of PBLs in B-CLL patients.
The current study was carried out on 15 B-CLL patients (5 males and 10 females), who were recently diagnosed and still untreated cases and 15 normal subjects (6 males and 9 females).
The study design included both clinical assessment, with special emphasis on lymph nodes, liver and spleen, routine hematological examinations, immunophenotyping and immunological studies.
Concerning clinical assessment, according to Rai classification, there was (≈7%) of B-CLL patients in stage 0, (20%) in stages I+II and (≈73%) in stages III+IV.
As regards immunological studies, PBLs were isolated from normal subjects and B-CLL patients and cultured in complete culture media without and with 1μM/ml Fludarabine for 48 hrs. Harvested cells were tested by flow cytometric techniques for the degree of apoptosis and IL-4 content using Annexin-V/PI staining and specific monoclonal IL-4 antibodies, respectively.
The results of our study can be summarized in:
1- The rate of in vitro spontaneous apoptosis of PBLs in B-CLL cells was nearly the same as in normal subjects. This may be attributed to absence of in vivo microenvironment as well as modulations in constitutive genetic defects of leukemic B-CLL cells when cultured in vitro.
2- Fludarabine- increased the rate of PBLs’ apoptosis in vitro in both B-CLL patients and normal subjects. However, the cytotoxic effect of Fludarabine was significantly higher in B-CLL patients than normal subjects.This suggested that B –CLL cells are more susceptible to Fludarabine than normal cells.
3- No correlations were found between in vitro spontaneous or Fludarabine induced apoptosis and age, sex, total leukocytic count or clinical stage.
4- Cytolasmic IL-4 content as well as percentage of IL-4+ve PBLs without Fludarabine were nearly the same both in normal subjects and B-CLL patients with no significant difference. This might be attributed to absence of in vivo activation signals as well as cell / cell and cell/ microenvironment interaction. In addition, in the current study IL-4 content was assessed without the addition of any cell stimulant.
5- Cytoplasmic content of IL-4 as well as the percentage of IL-4+ve PBLs were significantly increased in both normal subjects and B-CLL patients when PBLs were cultured with Fludarabine. However, no significant difference was observed when studied groups are compared to each other. Increased IL-4 content by Fludarabine is mostly due to increased rate of IL-4 synthesis. Such increase might influence the therapeutic effect of Fludarabine in B-CLL patients. So, the use of anti-IL-4 in combination with Fludarabine could help in avoidance of drug resistance.
6- No correlation was observed between the intracellular cytoplasmic IL-4 content and in vitro spontaneous or Fludarabine –induced apoptosis.
Accordingly, data of the current study suggested that Fludarabine induced apoptosis is mostly not mediated via modulation of IL-4 level. On the contrary, Fludarabine increased IL-4 content which may be one mechanism of drug resistance.