Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.

Curcumin has been shown to possess variety of biological functions including anti-tumor activity. The mechanism by which curcumin inhibit cell proliferation remains poorly understood. In the present report, we investigated the effect of curcumin on the activation of apoptotic pathway in T-cell acute lymphoblastic leukemia (T-ALL) malignant cells. Our data demonstrate that curcumin causes dose dependent suppression of proliferation in several T cell lines. Curcumin treatment causes the de-phosphorylation/inactivation of constitutively active AKT, FOXO transcription factor and GSK3. Curcumin also induces release of cytochrome c accompanied by activation of caspase-3 and PARP cleavage. In addition, zVAD-fmk, a universal inhibitor of caspases, prevents caspase-3 activation and abrogates cell death induced by curcumin treatment. Finally, treatment of T-ALL cells with curcumin down-regulated the expression of inhibitor of apoptosis protein (IAPs). Taken together, our finding suggest that curcumin suppresses constitutively activated targets of PI3'-kinase (AKT, FOXO and GSK3) in T cells leading to the inhibition of proliferation and induction of caspase-dependent apoptosis.

HLA-DPB1*0401 is associated with dominant protection against type 1 diabetes in the general Saudi population and in subjects with a high-risk DR/DQ haplotype.

The human leukocyte antigen (HLA) class II DQB1*0201/0202-DRB1*04 genotype has been identified as predisposing to type 1 diabetes [insulin-dependent diabetes mellitus (IDDM)] in the Saudi Arabian population (P = 0.0002; odds ratio = 0.67; 95% confidence interval = 0.009-0.381). In this study, we searched for a factor at the DPB1 locus by analysing DPB1 polymorphism using sequence-based typing in 86 Saudi IDDM patients and control subjects, all carrying the HLA-DRB1*04/DQB1*02 haplotype or the known susceptibility allele DQB1*0201/0202. Significant protection was conferred by DPB1*0401, which was observed in 17 of 50 control subjects (55%) and 2 of 36 IDDM patients (5%) with the DQB1*0201/0202 allele (P = 0.0012; odds ratio = 8.75; confidence interval = 1.72-59.70). Our data showing a high frequency of the DPB1*0401 allele even in the presence of the predisposing DQB1*02 allele in healthy subjects may indicate a protective effect of this combination of HLA alleles against type 1 diabetes. This finding supports the hypothesis that protective HLA class II genes can override the risk conferred by HLA-DQ susceptibility alleles. Further studies using larger cohorts of control subjects and patients should be undertaken to confirm this observation.

HLA class II sequence-based typing in normal Saudi individuals.

We have adopted a system that combines low resolution PCR-SSP followed by sequence-based typing (SBT) to analyze HLA-DRB1, -DPB1 and -DQB1 alleles in the Saudi population. The SBT method was used to identify HLA class II alleles in Saudis for the first time. Nineteen HLA-DRB1 alleles in currently recognized subtypes of the DRB locus were detected. DR1 and DR9 were not encountered. SBT did not detect diversity within the DR7 and DR10 alleles. Sixteen HLA-DQB1 and 10 HLA-DPB1 alleles were identified. This study represents the first molecular report on the HLA class II allele frequency in the population of Saudi Arabia.

Monitoring of donor/recipient T-cell engraftment kinetics in myeloablative allogeneic stem cell transplantation using short tandem repeat amplification from cell lysates.

Molecular monitoring of donor/recipient T-cell kinetics early post-transplant can provide clues to the immunological events that govern host-versus-graft reaction (HVGR) and graft versus-host-disease (GVHD). We have previously used fluorescence in situ hybridization (FISH) with X and Y probes to monitor recipient T (R-T) cell clearance early after myeloablative allogeneic stem cell transplantation (ASCT). We demonstrated that impaired clearance of residual host-T-cells in the early days post-transplant was associated with graft rejection, while enhanced clearance could be an indicator of increased donor anti-host alloreactivity and predictive of acute GVHD. Although FISH is the most accurate quantitative molecular tool for the determination of the exact donor/recipient-T-cell numbers at any time points post-transplant, it has the disadvantage of being limited to sex mismatched donor/recipient pairs. Our goal was to develop a molecular approach that, irrespective of gender, would be comparable to FISH in accurately determining host residual T-cell clearance after myeloablative conditioning for ASCT. We have genotyped DNA from cell lysates using polymerase chain reaction (PCR) amplification of short tandem repeats (STR) with fluorescently labeled oligonucleotide primers, and used the Genescan 672 software for accurate quantitative analysis of the amplified alleles. Here, we show that this approach allowed us to achieve in T-cells accurate quantitative analyses of amplified donor/recipient alleles in sex matched patients on days +5, +8 and +12 post-transplant, despite severe leukopenia.

The clinical significance of post-transplantation non-HLA antibodies in renal transplantation.

This study was undertaken to examine the clinical relevance of antibodies detected in the sera of patients following renal transplantation. The sera from 23 transplant recipients with acute rejection and 10 transplant recipients with diagnosed chronic rejection were tested against various epithelial, monocyte and endothelial cell lines (A549, HTB44, primary renal epithelial, U937 and Ea-hy 926). The test used for detecting binding antibodies was a simple, indirect immunofluorescence flow cytometric technique. The level of IgG antibodies directed against the test cell lines was examined in the sera of patients with mild or severe rejection and compared to those of patients showing no signs of rejection. Patients with chronic rejection were found to have increased levels of antibodies (IgG and IgM) when compared to patients with either end-stage renal failure or patients with stable post-transplant renal function. Antibodies detected by the present technique were directed against antigens found on all cell lines tested, and immunoblotting indicated that they were directed against non-HLA antigens. In conclusion, monitoring for the presence of such antibodies may provide a valuable prognostic indicator of graft rejection in renal transplant patients.

Characterization of donor-directed antibody class in the post-transplant period using flow cytometry in renal transplantation.

Over the past few years there has been increasing awareness of the importance of humoral mechanisms in the rejection of renal transplants. In this study we have monitored the development of antibodies directed against donor T and B lymphocytes using the sensitive flow cytometric technique. Forty-two cadaveric renal transplants were studied both before and for a maximum of 14 days after transplantation. Donor cells were separated from spleen on the day of transplantation and stored in liquid nitrogen until required. The dual colour flow cytometric assay was used to detect IgG or IgM directed against donor T or B lymphocytes. Using AB sera as controls, results were expressed as relative median fluorescence (RMF) and then correlated with the clinical performance of the grafts. Significant associations were found between the incidence of donor-directed antibodies and the development of clinical rejection. The magnitude of the rise in antibody levels was also related to graft performance. In patients showing severe graft rejection, high levels of antibodies of the IgG class developed before the clinical diagnosis of rejection was made. The routine use of this test allows the prediction of impending severe rejection to be made and may have important implications for immunosuppressive therapy.

Analysis of peripheral blood lymphocyte subsets in normal Saudi males by flow cytometry.

Lymphocyte immunophenotyping using flow cytometer has become an important tool for clinical patient management as well as for research and epidemiological studies. We examined the distribution of CD3 (all T cells), CD4 (T helper/inducer cells), CD8 (T suppressor/totoxic cells), CD16 (natural killer cells) and CD19 (B cells) in 150 healthy Saudi male blood donors using flows cytometry. The two-color labeled cells were analyzed by using the flow cytometer (FACScan, Becton-Dickinson, San Jose, California, USA) and the dual fluorescent subsets were discriminated by Simultest software. The distribution of T lymphocytes, B lymphocytes, and natural killer (NK) cells were similar to those reported in other populations as well as in normal Caucasian expatriate donors (all males) (n = 40) who were included in this study as controls. However, a significantly decreased CD4/CD8 ratio was observed in most Saudi blood donors. These lower ratios were due to decreased CD4 together with an increase in CD8 cells. Significant (P<0.00001) difference in CD4/CD8 ratio in our study may be due to environmental factors such as ultraviolet radiation and stress (heat) as well as some genetic factors.