Clinical Impact of Overexpression of FOXP3 and WT1 on Disease Outcome in Egyptian Acute Myeloid Leukemia Patients

Background: In the last decade, it has become clear that change of gene expression may alter the hematopoietic cell quiescent state and consequently play a major role in leukemogenesis. WT1 is known to be a player in acute myeloid leukemia (AML) and FOXP3 has a crucial role in regulating the immune response. Objectives: To evaluate the impact of overexpression of WT1and FOXP3 genes on clinical course in adult and pediatric AML patients in Egypt. Patients and methods: Bone marrow and peripheral blood samples were obtained from 97 de novo non M3 AML patients (63 adult and 34 pediatric). Real-time quantitative PCR was used to detect overexpression WT1 and FOXP3 genes. Patient follow up ranged from 0.2 to 39.0 months with a median of 5 months. Results: In the pediatric group; WT1 was significantly expressed with a high total leukocyte count median 50X109/L (p=0.018). In the adult group, WT1 had an adverse impact on complete remission induction, disease-free survival and overall survival (p=0.02, p=0.035, p=0.019 respectively). FOXP3 overexpression was associated with FAB subtypes AML M0 +M1 vs. M2, M4+M5 (p =0.039) and the presence of hepatomegaly (p=0.005). Conclusions: WT1 and FOXP3 overexpression has an adverse impact on clinical presentation, treatment response and survival of pediatric and adult Egyptian AML patients.     

Alanine Mutagenesis of the Primary Antigenic Escape Residue Cluster,C1, of Apical Membrane Antigen 1

Antibodies against apical membrane antigen 1 (AMA1) inhibit invasion of Plasmodium merozoites into red cells, and a large number of single nucleotide polymorphisms on AMA1 allow the parasite to escape inhibitory antibodies. The availability of a crystal structure makes it possible to test protein engineering strategies to develop a monovalent broadly reactive vaccine. Previously, we showed that a linear stretch of polymorphic residues (amino acids 187 to 207), localized within the C1 cluster on domain 1, conferred the highest level of escape from inhibitory antibodies, and these were termed antigenic escape residues (AER). Here we test the hypothesis that immunodampening the C1 AER will divert the immune system toward more conserved regions. We substituted seven C1 AER of the FVO strain Plasmodium falciparum AMA1 with alanine residues (ALA). The resulting ALA protein was less immunogenic than the native protein in rabbits. Anti-ALA antibodies contained a higher proportion of cross-reactive domain 2 and domain 3 antibodies and had higher avidity than anti-FVO. No overall enhancement of cross-reactive inhibitory activity was observed when anti-FVO and anti-ALA sera were compared for their ability to inhibit invasion. Alanine mutations at the C1 AER had shifted the immune response toward cross-strain-reactive epitopes that were noninhibitory, refuting the hypothesis but confirming the importance of the C1 cluster as an inhibitory epitope. We further demonstrate that naturally occurring polymorphisms that fall within the C1 cluster can predict escape from cross-strain invasion inhibition, reinforcing the importance of the C1 cluster genotype for antigenic categorization and allelic shift analyses in future phase 2b trials.The merozoite stage of Plasmodium falciparum is a highly specialized form of the parasite that selectively invades human red blood cells. Although the exact mechanism of invasion is still under investigation, the expression of apical membrane antigen 1 (AMA1) appears to be an absolute requirement for successful invasion (31). Monoclonal and polyclonal antibodies against AMA1 inhibit invasion in vitro, and immunization with recombinant AMA1 protects against live parasite challenge in animal models of malaria (10a, 18, 28).P. falciparum AMA1 vaccines based on two laboratory strains, 3D7 and FVO, are being studied for efficacy in human trials (23, 27, 30). One of the major concerns in further development of the AMA1 vaccine is that ∼10% of its 622 amino acids are polymorphic (2). Strain specificity of vaccine-induced AMA1 antibodies has been observed by enzyme-linked immunosorbent assay (ELISA) and in a functional assay of parasite growth and invasion inhibition (growth inhibition assay [GIA]) (15, 24). Allelic replacement experiments show that sequence polymorphism within AMA1 causes antigenic escape (14), and the extent of escape correlates with sequence distance between the vaccine and target strain (18).The crystal structure of AMA1 shows that it contains two PAN domains, with loops extending outwards from its central core (1, 22). The loops contain the majority of the polymorphic residues of AMA1 and surround a highly conserved hydrophobic trough. Residues within the trough have been implicated in the binding of AMA1 to AMA1-associated proteins on the merozoite (5). The location of the polymorphic loops surrounding the trough is highly suggestive of its role of providing diversity to a functional region of AMA1, preventing the binding of inhibitory antibodies. Using chimeric proteins to specifically deplete strain-specific antibodies against 3D7 strain AMA1, in a growth inhibition assay (GIA), we showed that certain polymorphic sites conferred escape upon the FVO strain parasite from invasion inhibitory anti-3D7 AMA1 antibodies. These polymorphic residues were termed “antigenic escape residues” (AER). The majority of AER in the 3D7-FVO model mapped to domain 1 (10), and within domain 1, the highest escape per residue was conferred by 7 polymorphic sites located on a linear stretch of sequence between residues 187 and 207. This polymorphic cluster was termed the C1′ cluster (10). It contains the three most polymorphic residues of AMA1, as follows: residue 187 is located on loop Ic, and residues 197 and 200 are located on the adjacent C1-L loop (also termed loop Id).Given that the polymorphic residues within the C1 cluster are the primary determinants of strain specificity of AMA1, it is likely that protein engineering strategies targeted to C1 AER could be used to modulate the cross-reactivity of AMA1 antibodies. We hypothesized that if the immunogenicity of the C1 AER could be reduced, this might result in an increased antibody response to non-strain-specific cross-reactive epitopes. In this study, seven 3D7-FVO polymorphic differences within the C1 cluster were replaced on the FVO AMA1 protein with alanine residues. The 3D7-FVO escape model was then used to study the effect of alanine mutagenesis on the quantity and quality of the induced antibodies. This strategy resulted in a measurable shift in the immune response away from the C1 region but did not enhance the generation of cross-strain-reactive antibodies that were inhibitory to parasite invasion.     

Methylation-mediated silencing and tumour suppressive function of <Emphasis Type="Italic">hsa-miR-124</Emphasis> in cervical cancer

Background  

A substantial number of microRNAs (miRNAs) is subject to epigenetic silencing in cancer. Although epigenetic silencing of tumour suppressor genes is an important feature of cervical cancer, little is known about epigenetic silencing of miRNAs. Since DNA methylation-based silencing of hsa-miR-124 occurs in various human cancers, we studied the frequency and functional effects of hsa-miR-124 methylation in cervical carcinogenesis.     

Tumor necrosis factor-α and interleukin-6 in cirrhotic patients with spontaneous bacterial peritonitis

AIM: To evaluate the role of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in cirrhotic patients who have hepatic and renal impairment with spontaneous bacterial peritonitis (SBP).METHODS: We prospectively studied 120 cirrhotic patients with SBP and 80 cirrhotic patients with sterile ascitic fluid. They included 144 males and 56 females with ages ranging between 34 and 62 years. The diagnosis of cirrhosis was established by clinical and laboratory criteria that did not require histological confirmation. The severity of underlying liver disease was evaluated using Pugh’s modification of Child’s criteria (Child-Pugh scores). Ascitic fluid was sent to the laboratory for cell count, culture, sensitivity testing, and measurement of chemical elements (i.e., albumin, glucose). Specimens were inoculated into aerobic and anaerobic blood culture bottles. Serum and ascitic fluid were also collected in sterile tubes at study entry (before the initiation of antibiotic treatment) and 48 h later. Assays for TNF-α and IL-6 in the serum and ascitic fluid were performed with an immunoenzymometric assay using manufacture’s instructions.RESULTS: Cytokine levels in serum and ascitic fluid were significantly higher in the patients with SBP. (plasma TNF-α: 135.35 ng/mL ± 11.21 ng/mL vs 92.86 ng/mL ± 17.56 ng/mL, P < 0.001; plasma IL-6: 32.30 pg/mL ± 7.07 pg/mL vs 12.11 pg/mL ± 6.53 pg/mL, P < 0.001; ascitic fluid TNF-α: 647.54 ± 107.11 ng/mL vs 238.43 ng/mL ± 65.42 ng/mL, P < 0.001); ascitic fluid IL-6: 132.84 ng/mL ± 34.13 vs 40.41 ± 12.85 pg/mL, P < 0.001). About 48 (40%) cirrhotic patients with SBP developed renal and hepatic impairment and showed significantly higher plasma and ascitic fluid cytokine levels at diagnosis of infection. [(plasma TNF-α: 176.58 ± 17.84 vs 135.35 ± 11.21 ng/mL) (P < 0.001) and (IL-6: 57.83 ± 7.85 vs 32.30 ± 7.07 pg/mL) (P < 0.001); ascitic fluid TNF-α: 958.39 ± 135.72 vs 647.54 ± 107.11 ng/mL, (P < 0.001), ascitic fluid IL-6: 654.74 ± 97.43 vs 132.84 ± 34.13 pg/mL, (P < 0.001)]. Twenty nine patients (60.4%) with SBP and renal impairment died whereas, only four patients (5.55%) with SBP but without renal impairment died from gastrointestinal hemorrhage (P < 0.0005).CONCLUSION: It appears that TNF-α production may enhance liver cell injury and lead to renal impairment. This correlated well with the poor prognosis and significantly increased mortality associated with SBP in cirrhotic patients.