Absolute CD4 count and percentage values among Libyan patients with HIV by single-platform flow cytometry.

BACKGROUND: Single-platform flow cytometry technology together with CD45-gating is becoming the method of choice for absolute CD4 T cell enumeration. Immunological assessment of HIV patients by monitoring CD4 can provide valuable information on antiviral treatment response and disease progression. METHODS: A total of 97 HIV-positive individuals were recruited from 2 hospitals in Tripoli, Libya, and 14 healthy blood donors. The HIV-infected individuals were classified by CD4+ count into HIV-positive (>200 cells/µL) or AIDS (≤200 cells/µL) groups. CD4+ and CD8+ cell counts were determined and compared among the groups and with similar published data. RESULTS: The mean ± SD CD4+ cell counts were 1106 ± 442.8 cells/µL in healthy individuals, 460 ± 219.7 cells/µL in the HIV-positive group, and 78 ± 64.3 cells/µL in the AIDS group. The mean ± SD CD4+/CD8+ ratio was 1.6 ± 0.58, 0.4 ± 0.22, and 0.1 ± 0.1, respectively. CD4+ counts in Libyan healthy adults might be higher than those reported in several studies in other regions, whereas CD4+ counts in Libyan AIDS patients seem lower. CONCLUSION: Reference values for T lymphocyte counts in Libyan healthy individuals should be investigated more extensively, and the reasons why Libyan AIDS patients seem to have such lower CD4+ counts should be examined.

Flow cytometric detection of leukemic blasts in Libyan pediatric patients with acute lymphoblastic leukemia.

The diagnosis of acute lymphoblastic leukemia (ALL), which is the most common type of cancer in children, has become more accurate with the use of flow cytometry. Here, this technology was used to immunophenotype leukemic cells in peripheral blood samples from Libyan pediatric ALL patients. We recruited 152 newly diagnosed patients at Tripoli Medical Center (Tripoli, Libya) by morphological examination of blood and bone marrow. Twenty-three surface and cytoplasmic antigen markers were used to characterize B and T cells in circulating blood cells by four-color flow cytometry. Six children (3.9%) turned out to have biphenotypic acute leukemia, 88 (57.9%) had B ALL, and 58 (38.1%) had T ALL. There were 68 cases of pro-B ALL CD10-positive (44.7%), 8 cases of pro-B ALL CD10-negative (5.2%), 6 cases of pre-B ALL (3.9%), and 6 of mature-B ALL (3.9%). CD13 was the most commonly expressed myeloid antigen in ALL. We present immunophenotypic data for the first time describing ALL cases in Libya. The reported results indicate that the most common subtype was pro-B ALL, and the frequency of T-ALL subtype was higher compared to previous studies. Six cases were positive for both myeloid and B lymphoid markers. Our findings may provide the basis for future studies to correlate immunophenotypic profile and genetic characteristics with treatment response among ALL patients.

Rituximab increases the cytotoxicities of vincristine and hydroxyurea through caspase-dependent and caspase-independent cell death, respectively.

In the treatment of B cell non-Hodgkin's lymphoma, rituximab is used in combination with different chemotherapeutics to improve its efficacy, but the mechanisms involved are not fully understood. The authors examined the mechanism by which rituximab combined with hydroxyurea or vincristine induces cell death in the human Burkitt's lymphoma Ramos cell line. Cell death was analyzed by phosphatidylserine exposure, caspase activation, and mitochondrial membrane changes. Their results indicate that the cell death initiated by the combination of rituximab and hydroxyurea is caspase-independent. In contrast, preincubation of the cells with the same concentrations of caspase inhibitors used with hydroxyurea eliminated the synergistic effect of the rituximab and vincristine combination. This was confirmed by the presence of the active fragment of caspase-3 in vincristine-treated cells. These preliminary results demonstrate that rituximab can activate different downstream signals to induce direct cell effects. Furthermore, the findings support the important role of mitochondria in the regulation of both pathways.

Rituximab: modes of action, remaining dispute and future perspective.

Less than two decades ago, immunotherapy joined chemotherapy and radiotherapy as an effective approach for the treatment of cancer. The anti-CD20 monoclonal antibody, rituximab, is now used to treat almost all types of non-Hodgkin's B-cell lymphomas, and it could be useful in the treatment of other diseases with B-cell involvement. Upon binding, rituximab induces death of the target cells. It seems to act not only by activating immune system defense mechanisms such as complement-dependent and antibody-dependent cellular cytotoxicity, but also by inducing direct cell death. In this paper, we review current knowledge on rituximab mechanisms of action, with particular attention to its direct effects, and also highlight potential future avenues of research.

Rituximab synergizes with hydroxyurea or vincristine in the killing of Ramos Burkitt's lymphoma B cell line.

Rituximab is an effective immunotherapy for CD20-positive B-cell non-Hodgkin's lymphoma. However, some patients show resistance, particularly those suffering from more aggressive lymphoma types, such as Burkitt's lymphoma. Hence, Rituximab is commonly combined with several chemotherapeutic drugs. With a view to reduce the number of such drugs, we examined the effect of combining Rituximab individually with hydroxyurea, vincristine, or etoposide on the killing of Ramos Burkitt lymphoma cell line type I. Cell death was examined by using Annexin-V/propidium iodide staining. Combining Rituximab with hydroxyurea or vincristine resulted in a synergistic effect, whereas combining it with etoposide resulted in a subadditive effect. In single treatments, the percentage of cell death ranged from 23% (Rituximab) to 36% (hydroxyurea). Combining Rituximab with hydroxyurea or vincristine resulted in a synergistic effect (83% and 74% killing, respectively). In contrast, only a subadditive effect was noticed with etoposide (36%). We conclude that the synergistic effect of Rituximab with hydroxyurea or vincristine is worthy of further study, and that further in vitro screening of chemotherapeutics might identify chemo-immunotherapeutic combinations that are effective in vivo but less toxic than currently used regimens.

Hydroxyurea potentiates the caspase-independent killing of B-cell lines by rituximab and GA101.

Anti-CD20 monoclonal antibodies have revolutionized the treatment of non-Hodgkin's lymphoma over the last decade. Unfortunately, a significant number of patients treated by these antibodies exhibit innate or acquired antibody resistance and fail to respond to treatment. Strategies to improve antibody function and overcome resistance include the development of new "engineered" antibodies and the use of new drug combination therapies. In this report, we show that the antimetabolite hydroxyurea significantly enhances the ability of two therapeutic monoclonal antibodies to directly kill some human B-cells. The two anti-CD20 antibodies studied were a clinically well-established type 1 therapeutic antibody, namely rituximab and GA101, an antibody representing the new breed of type 2 glycoengineered monoclonals. Hydroxyurea specifically enhanced the direct caspase-independent killing pathway of both of these antibodies as exemplified by the resistance to broad spectrum caspase inhibitors, lack of internucleosomal DNA laddering, and lack of activation of caspases 3, 8, and 9. Both rituximab and GA101 appear to preferentially kill cells in the G0/G1 cell cycle phase. One of the many reported effects of hydroxyurea is cell arrest in this phase. Arresting antibody-sensitive cells in this stage of the cell cycle by means other than hydroxurea also sensitized the cells to caspase-independent antibody-mediated death, suggesting that the potentiating effect of hydroxyurea may be mediated via its effects upon the cell cycle. The possible combination of hydroxyurea and anti-CD20 monoclonal antibodies may offer new possibilities for combination therapies in the clinic.

Depletion of peripheral blood B cells with Rituximab and phenotype characterization of the recovering population in a patient with follicular lymphoma.

The monoclonal antibody Rituximab is useful for treatment of patients with B-cell non-Hodgkin's lymphoma. We phenotypically analyzed reconstitution of peripheral B cells in a male patient with follicular lymphoma following their depletion with Rituximab. CD19+ and CD20+ B cell counts in peripheral blood decreased rapidly following Rituximab treatment. Six months after the end of treatment, a few CD19+ B cells were detected in peripheral blood. These cells had a naive B cell phenotype (IgD+, CD27-) and they expressed high levels of CD38 and CD24, which show that the B cell pool was repopulated mainly with immature, naive B cells.