Manal Mohamed Makhlouf

Hematopoietic stem cell mobilization into the peripheral circulation in patients with chronic liver diseases

The present study was aimed to investigate and compare the kinetics of bone marrow‐derived hematopoietic stem cells (BMHSC) migration in the peripheral blood and liver in response to liver injury in patients with chronic liver disease (CLD).

Detection of XRCC1 gene polymorphisms in Egyptian patients with acute myeloid leukemia

XRCC1 is a polymorphic gene belonging to one of the major deoxyribonucleic acid (DNA) repair pathways. XRCC1 is involved in base excision repair (BER) and the repair of single-strand breaks. Several variants of XRCC1 have been described, including one affecting codon 399 in exon 10 that results in an arginine (Arg) to glutamine (Gln) substitution and one affecting codon 194 in exon 6 that results in an Arg to tryptophan (Trp) substitution. The aim of this study was to determine the presence of these polymorphisms in the Egyptian population and to define their role in modulating susceptibility to development of acute myeloblastic leukemia (AML) using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique in 40 de novo AML patients and 20 controls. The risk of development of AML was found to be significantly increased when variant XRCC1-399 (Arg/Gln) is present (P value 0.025). Moreover, the risk of AML development was found to be significantly increased when variant XRCC1-194 (Arg/Trp) is present (P value 0.002), whereas the risk of AML development is even higher when both variants XRCC1-Arg-399 Gln and Arg-194 Trp alleles are present (odds ratio [OR] 6.15 and 4.00 and 95% CI 1.88–20.05 and 1.13–14.08, respectively), presumably because an increase in DNA damage significantly increases the risk of development of AML, and the phenotypes, as a result, interact to increase this risk. These results strongly suggest that BER pathway, notably XRCC1, is important in the pathogenesis of de novo AML.

Genetic polymorphism of microsomal epoxide hydrolase enzyme gene in preeclamptic females.

Introduction:Microsomal epoxide hydrolase enzyme is involved in xenobiotics detoxification. It catalyzes the phase I hydrolysis of epoxides and plays a role in the detoxification processes and in the metabolism of endogenous and exogenous compounds. Preeclampsia, which is one of the most serious complications of pregnancy, may be due to an imbalance between these compounds, such as lipid peroxides and oxygen-free radicals and detoxifying and scavenging substances. Two variants of human epoxide hydrolase enzyme with different enzyme activity have been described; exon 3 polymorphism is associated with lower enzyme activity whereas exon 4 polymorphism is associated with higher activity. The authors tried to investigate the association between these genetic polymorphisms and preeclampsia. Method:Thirty preeclamptic females together with 30 normal pregnant females as controls were included in the study. Genotyping for exons 3 and 4 of microsomal epoxide hydrolase enzyme was done by polymerase chain reaction–restriction fragment length polymorphism. Results:There was no statistical significant difference in the distribution of exon 3 genotype between cases and controls (P = 0.4); on the other hand, a highly statistical significant difference was found between cases and controls as regard exon 4 genotype (P = 0.002). Conclusion:There may be an association between epoxide hydrolase enzyme polymorphism and the risk of preeclampsia.

Chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in vitro

Different therapeutic techniques have been developed for regeneration of articular cartilage injuries, but none has provided an optimal solution to their treatment. Human umbilical cord blood‐mesenchymal Stem Cells (HUCB‐MSCs) have been considered as promising alternative cell source for cartilage repair. Objectives: Examining the success rate of MSCs isolation from HUCB as well as chondrogenic differentiation potential of HUCB‐MSCs in vitro. Materials and methods: 32 UCB samples were collected, in addition to 5 bone marrow (BM) and 5 peripheral blood (PB) samples, taken as reference controls. Samples were used for mononuclear cells isolation from which MSCs were expanded under complete aseptic conditions, were verified morphologically and through the presence of CD44 and CD105, and absence of CD34. Results: Success rate of UCB‐MSCs isolation was (25%), a rate that was lower than those of PB (40%) and BM (80%). Accordingly, certain input parameters have been recommended for successful MSCs isolation from UCB. On selecting samples in which recommended parameters were fulfilled, success rate was increased to 72%. This was together with providing optimal experiment conditions; mainly type of expansion medium, success rate reached 80%. Then, successfully expanded MSCs were subjected to chondrogenic differentiation by culturing in pelleted micromass system in presence of transforming growth factor beta‐1 and chondrogenic medium devoid of fetal bovine serum to evaluate their ability to undergo chondrogenesis. Differentiation was verified microscopically using special stains, and proved by reverse transcriptase‐polymerase chain reaction for expression of aggrecan and collagen II genes. In conclusion, in vitro differentiation into chondrocytes is possible from HUCB‐MSCs. Microsc. Res. Tech. 78:667–675, 2015.

Expression of IL4 (VNTR intron 3) and IL10 (-627) Genes Polymorphisms in Childhood Immune Thrombocytopenic Purpura

OBJECTIVE Immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder caused by the production of antiplatelet antibodies. These autoantibodies opsonize platelets for splenic clearance, resulting in low levels of circulating platelets. Interleukin 4 (IL4) and interleukin 10 (IL10) are important immunoregulatory cytokines mainly produced by macrophages, monocytes, T cells, B cells, and mast cells. Our study was aimed at detecting the frequency of IL4 (VNTR intron 3) and IL 10 (-627) gene polymorphisms in Egyptian ITP children as genetic markers for ITP risk and clarifying their possible role in the pathogenesis of ITP as well as their correlation with the clinical presentation and laboratory data. METHODS IL4 (VNTR intron 3) and IL10 (-627) gene polymorphisms were studied in 70 ITP patients and 50 age- and sex-matched healthy controls using polymerase chain reaction-restriction fragment length polymorphism assay (PCR-RFLP). RESULTS IL4 RP2 and IL10 A alleles were detected more frequently among ITP patients compared to controls. A statistically significant difference was observed in IL10 and IL4 gene polymorphism distribution between acute and chronic ITP patients, with higher A allele and RP2 allele among chronic ITP patients versus acute ITP patients. Combined polymorphisms of IL4 and IL10 genes were associated with greater risk of ITP. CONCLUSION IL4 and IL10 gene polymorphisms may contribute to susceptibility for ITP in children.

TAp73 and ΔNp73 relative expression in Egyptian patients with lymphoid neoplasms.

Background and aims The p73 gene has different isoforms with opposing anti- and pro-apoptotic functions. The pro-apoptotic activities are inhibited by overexpression of the dominant ΔNp73 isoform. The aim of this study was to detect the expression of the TAp73 and ΔNp73 isoforms in Egyptian patients with malignant lymphoid neoplasms. Their expression was analyzed by quantitative RT-PCR. Patients and Methods The study included 30 B-NHL patients, 24 T-NHL patients, 16 ALL patients, 18 CLL patients, 22 patients with reactive lymphoid hyperplasia, and 6 healthy control subjects. Results ALL and CLL patients expressed both isoforms at higher levels compared to lymphoma patients. Higher expression of TAp73 was found in both B-NHL and T-NHL (around 4-fold and 16-fold, respectively) in comparison to ΔNp73 (2-fold and 14-fold, respectively). In CLL patients both isoforms showed higher expression levels in comparison to normal peripheral blood lymphocytes controls: nearly 27-fold for TAp73 and 233-fold for ΔNp73. All ALL patients showed higher expression of both studied isoforms than controls (9-fold for TAp73 and 386-fold for ΔNp73). The highest ΔNp73 expression along with a higher ΔNp73/TAp73 ratio (67-fold) was found in ALL patients compared with CLL patients (21-fold). Conclusions A considerable number of lymphoma patients lacked the expression of either or both isoforms, while all lymphoid leukemia patients expressed both isoforms. The expression pattern differences of p73 isoforms may reflect differences in the biology of these malignancies.

Assessment of interleukin 28B genotype as a predictor of response to combined therapy with pegylated interferon plus ribavirin in HCV infected Egyptian patients

BACKGROUND AND AIM Single nucleotide polymorphisms (SNPs) of interleukin 28B (IL28B) gene is associated with spontaneous clearance and variable response to combined therapy with pegylated interferon (PEG-IFN) and ribavirin (RBV) in chronic hepatitis C virus (HCV) infected patients. This study aimed at assessing the value of IL28B rs8099917 gene polymorphism in predicting sustained virological response (SVR) among HCV infected Egyptian patients treated with PEG-IFN and RBV. METHODS Our study was conducted on 153 chronic HCV infected patients treated with PEG-IFN and RBV. Genotyping of rs8099917 near the IL-28B gene was performed by Real Time PCR using Taq-Man probe assay. RESULTS The overall SVR was achieved in 49.6% of patients. Patients with TT genotype showed significantly higher SVR rate than minor allele (TG/GG) carriers (74% vs. 26%, P=0.004). Logistic regression analysis revealed that TT carriers had 2.8 higher chance for SVR achievement than G allele carriers TG/GG (OR=2.8, 95% CI=1.4-5.6, P=0.004). Younger age, male sex and low activity grading were significant predictors of SVR (P=0.003, P=<0.001 and P<0.001 respectively). High pretreatment AST levels and advanced liver fibrosis were negative predictors of SVR (P=0.04 and P<0.001 respectively). CONCLUSION IL28B genotype is a significant pre-treatment predictor of response to PEG-IFN/RBV in HCV infected Egyptian patients.

The clinical relevance and prognostic significance of microsomal epoxide hydrolase gene polymorphisms and their susceptibility to acquired aplastic anemia: an Egyptian study.

Abstract Background: Microsomal epoxide hydrolase enzyme (mEPHX) is involved in xenobiotics detoxification. Two variants of mEPHX, Tyr113His and His139Arg, have been described. Both may lead to acquired aplastic anemia (AA). Objectives: Assessing mEPHX genetic polymorphisms and detecting their impact on susceptibility and prognosis in Egyptian AA patients. Participants and methods: mEPHX 113 and 139 genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 100 patients with AA and 100 control subjects. Results: Both mEPHX Tyr113His and His139Arg gene polymorphisms were associated with increased risk of developing AA, and have a significant impact of bad prognosis (p value < 0.01). Conclusions: These mEPHX gene polymorphisms can be considered as risk factors and predictive molecular markers for prognosis in AA patients.

Enumeration, isolation, and electron microscopic study of hematopoietic stem cells in Egyptian patients with chronic liver diseases

The contribution of hematopoietic stem cells (HSCs) to liver regeneration in different forms of liver injury remains debatable. Many studies tried to verify whether various liver lesions can activate bone marrow by mobilizing peripheral blood HSCs (CD34/CD133+cells) putatively able to induce liver repopulation. The aim of this work was to determine the degree of mobilization of BM-derived HSCs into the peripheral blood of chronic hepatic affection patients and correlating it with various grades of liver damage. This study was conducted on 30 patients with Child A, B, and C grades of chronic liver disease (ten patients for each stage) as well as ten age- and sex-matched normal healthy subjects were enrolled as a control group. The percent of circulating HSCs was determined by flow cytometry. Also, the isolation of such cells was done by magnetic cell-sorting technique for further ultrastructural assessment by transmission electron microscopy (TEM). Our study revealed that chronic liver disease patients compared to healthy control group, exhibited insignificant difference in the percentage of circulating CD133+ cells. Regarding the level of CD34+ cells, a significant increase was found between Child A chronic liver disease patients and the control group (p = 0.02). However, an insignificant difference was found between Child B and C patients and control group or between each other (p > 0.05). The ultrastructural characteristics of isolated cells were compared in healthy subjects and hepatic patients, the cells were similar in appearance in both groups with no evidence of structural changes. TEM analysis revealed typical features of immaturity. Our study showed that chronic lesions of any degree of severity did not evoke bone marrow to mobilize HSCs into the circulation.