Taghrid Gaafar

Evaluation of cytokines in follicular fluid and their effect on fertilization and pregnancy outcome.

Cytokines in follicular fluid (FF) are important for reproduction as they modulate oocyte maturation and ovulation which influence subsequent fertilization, development of early embryo and potential for implantation. We evaluated FF cytokines in women who underwent intracytoplasmic sperm injection (ICSI) and their association with fertilized oocytes, embryo quality and pregnancy outcome. FF belonging to 38 patients including 18 polycystic ovary (PCO) and 20 male/unexplained infertility patients were investigated for granulocyte colony stimulating factor (G-CSF), regulated upon activation normal T cell expressed and presumably secreted (RANTES), tumour necrosis factor (TNFα), interferon gamma (IFNγ) and interleukins (IL-4 and IL-2) by bead-based sandwich immunoassay. Our findings revealed that on the day of oocyte retrieval, G-CSF was positively correlated with the number of fertilized oocytes, while TNFα detection was associated with reduced number of fertilized oocytes. Only G-CSF showed significant positive effect to the pregnancy outcome although the cytokines studied were not associated with embryo quality. PCO as the cause of infertility did not show an association with cytokines in FF. The functions of cytokines in reproduction are likely to be complex, and cytokine evaluation may offer insight to the understanding of the mechanisms leading to success or failure of assisted reproduction.

Differential responsiveness of humans with early-stage schistosomiasis haematobium to Schistosoma haematobium soluble adult-worm and egg antigens

Abstract Schoolchildren (7–8 years old) infected with Schistosoma haematobium were tested for lymphocyte proliferative responses, in vitro granuloma formation (IVGF), and cytokine release in T-cell Western assays and for serum antibody reactivity by enzyme-linked immunosorbent assay (ELISA) and immunoblotting against S. haematobium soluble adult-worm (SAWA) and egg (SEA) antigens. The lymphoproliferative response rate of individual subjects against 10 SAWA and 15 SEA electroseparated bands ranged from 0 to 33 % and from 11 to 66 %, respectively. The SAWA bands essentially failed to elicit significant IVGF, in contrast to the SEA bands, all of which were capable of inducing IVGF from peripheral blood mononuclear cells (PBMC) of 30–80 % of individual donors. The exclusive ability of SEA bands to induce IVGF could not be attributed to selective release of interleukin 2 (IL-2), IL-4, or interferon-gamma, as SEA and SAWA bands were capable of eliciting release of a similar array of cytokines in supernatants of 4-day PBMC cultures. The antibody response to SEA was stronger than that to SAWA, yet the proportion of SAWA bands binding humoral antibodies of individual donors was significantly larger than that observed for SEA. The study thus suggests that humans with early-stage S. haematobium infection respond poorly to SAWA but mount strong cellular immune responses to SEA that result in granuloma and antibody formation.

Identification ofSchistosoma haematobium soluble egg antigens that elicit human granuloma formation in vitro

Schistosoma haematobium soluble egg antigens (SH SEAs) induce intense granulomas in human hosts that often culminate in severe disease. In an attempt to identify the SH SEA fractions that are responsible for pathology, we combined T-cell Western blotting and an in vitro model of granuloma formation. Whole SH SEAs were dotted onto nitrocellulose pieces or were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred onto nitrocellulose paper. Horizontal strips bearing the separated antigens were solubilized in dimethylsulfoxide and precipitated in carbonate/bicarbonate buffer. Antigen-free and antigen-bearing particles were used to stimulate peripheral blood mononuclear cells (PBMCs) obtained fromS. haematobium-infected patients and sex- and agematched healthy controls to form granulomas in vitro. Whole SH SEA-bearing nitrocellulose particles elicited in vitro formation of granulomas by PBMCs from infected donors. The response was similar in sensitivity, specificity, and reproducibility to that evoked by SH SEA-bound polyacrylamide beads. The results obtained in samples from 30 patients and 10 controls tested with SH SEA-separated fractions revealed that SEA bands of 84 000, 63 000, 57 000, 55 000, 40 000, 30 000, and 28 000 Da elicited in vitro granuloma reactions by PBMCs of almost all infected patients. Conversely, separated soluble adult-worm antigens failed to stimulate PBMCs of infected patients to form granulomas. This study is the first to identify the SH SEA fractions that evok in vitro granuloma formation and represents an initial step toward the development of an anti-urinary schistosomiasis pathology vaccine.

Immunomodulatory effects of umbilical cord-derived mesenchymal stem cells

Umbilical cord blood (UCB) is of great interest as a source of stem cells for use in cellular therapies. The immunomodulatory effect of mesenchymal stem cells (MSCs) originating from bone marrow, adipose tissue and amniotic membrane has previously been reported. In this study, MSCs were isolated from UCB with the aim of evaluating their immunomodulatory effects on proliferation of PB lymphocytes by two different techniques; namely, 5‐bromo‐2‐deoxyuridine ELISA and a carboxy fluorescein diacetate succinimidyl ester flow cytometric technique. MSCs were isolated from UCB, propagated until Passage four, and then characterized for cell surface markers by flow cytometry and ability to differentiate towards osteocytes and adipocytes. Immunosuppressive effects on PB lymphocytes were examined by co‐culturing mitomycin C‐treated UCB MSCs with mitogen‐stimulated lymphocytes for 72 hr. Thereafter, proliferation of lymphocytes was detected by CFSE flow cytometry and colorimetric ELISA. The titers of cytokines in cell culture supernatant were also assayed to clarify possible mechanisms of immunomodulation. UCB MSCs suppressed mitogen‐stimulated lymphocyte proliferation, which occurs via both cell‐cell contact and cytokine secretion. Titers of transforming growth factor beta and IL 10 increased, whereas that of IFN‐γ decreased in the supernatants of co‐cultures. Thus, UCB MSCs suppress the proliferation of mitogen‐stimulated lymphocytes. However further in vivo studies are required to fully evaluate the immunomodulatory effects of UCB MSCs.

The T H 17/Treg Imbalance in Rheumatoid Arthritis and Relation to Disease Activity

Objectives: T regulatory cells (Treg) and proinflammatory TH17 cells are newly identified T lymphocyte subsets, which have significant effects on autoimmunity and inflammation. Tregs play key role in the maintenance of self-tolerance. TH17/Treg disturbed balance has been reported to contribute to several autoimmune diseases.Our aim was to assess the Treg/TH17 pattern and TH17 related cytokines, in peripheral blood of Egyptian rheumatoid arthritis patients, and their relation to disease activity score (DAS). Analyzing IL17A and IL23 as indicators of TH17 function, was to study the effect of TH17 in RA.Methods: 100 Egyptian rheumatoid arthritis patients, divided into Group I (14), Group II (48), Group III (38), with low 5.1, activity DAS score respectively; and 50 healthy age and sex matched controls were enrolled. Peripheral blood TH17 and Treg (CD4+CD25+Foxp3+) frequencies were analyzed by flowcytometry, and the serum levels of interleukins (IL17A), (IL23) were determined by ELISA.Results: Active RA patients (groups II and III) revealed an obvious increase in peripheral TH17 frequencies, and levels of TH17-related cytokines, and a significant decrease in Treg (CD4+CD25+Foxp3+) frequencies in group III, when compared to healthy controls. TH17/Treg ratios were positively correlated with serum concentrations of IL17A and IL23 cytokines. Frequencies and levels showed statistical significant correlation with DAS scoring.Conclusions: Our study indicated that TH17/Treg balance was disturbed in peripheral blood of RA patients, leading to an increase of proinflammatory cytokines, correlating with DAS, and suggesting an important role in the development of RA.

Cardioprotective Effects of Wharton Jelly Derived Mesenchymal Stem Cell Transplantation in a Rodent Model of Myocardial Injury

Background Whartons jelly-derived mesenchymal stem cells are a valuable alternative source that possess multipotent properties, easy to obtain and available in large scale compared to BMMSCs. We investigated the possibility of cardiac function improvement post isoproterenol induced cardiac injury in a rat model following human WJMSCs transplantation. Materials and Methods MSCs were extracted and cultured from cord WJ, characterized by morphology, Immunophenotyping and differentiation to osteoblast and adipocytes. WJMSCs were labeled with PKH2 linker dye. Wistar rats were divided into control group, ISO group (injected with 2 doses of isoproterenol) to induce myocardial injury and ISO group transplanted with labelled WJMSCs. ECG, electrocardiographic patterns, cardiac marker enzymes, tracing of labeled MSCs and immunohistochemical analysis of myocardial cryosections were studied. Results and Conclusions WJ derived MSCs were expanded for more than 14 passages while maintaining their undifferentiated state, were positive for MSC markers and were able to differentiate into adipocyte and osteoblast. We demonstrated that intravenously administered WJMSCs were capable of homing predominently in the ischemic myocardium. Cardiac markers were positively altered in stem cell treated group compared to ISO group. ECG and ECHO changes were improved with higher survival rate. WJMSCs could differentiate into cardiac-like cells (positive for cardiac specific proteins) in vivo. WJMSCs infusion promoted cardiac protection and reduced mortality, emphasizing a promising therapeutic role for myocardial insufficiency.

The impact of growth factors on human induced pluripotent stem cells differentiation into cardiomyocytes

ABSTRACT Human induced pluripotent stem cells (hiPSCs) act as a promising therapeutic alternative for cardiovascular diseases. They yield a large number of functional cardiomyocytes (CMs) from autologous cell sources without ethical or immunological problems. However, significant limitations still remain in terms of line‐to‐line variability in CM yield and reproducibility. Aim: To efficiently enhance NP0040 hiPSCs differentiation into CMs. Main methods: Following a standard cardiac differentiation protocol using small molecules targeting the canonical Wnt signaling, growth factors (BMP4 and FGF2) and ascorbic acid were added further in order to increase the cardiac differentiation efficiency. All cultures were conducted in serum‐free, feeder‐free monolayer system followed by lactate purification. Key findings: Using NP0040 hiPSCs, the CM yield resulting from modulation of the Wnt signaling pathway alone was inefficient compared to previous studies while the addition of BMP4, FGF2 and ascorbic acid resulted in enhanced cardiac differentiation outcome. The later resulted in a high yield (up to 92%) of cardiac troponin‐T (cTnT) + CMs contracting spontaneously as organized sheets in 15 independent experiments. They were validated structurally and functionally using immunofluorescent staining for sarcomeric &agr;‐actinin, cTnT, MLC2v and Connexin 43. Reverse‐transcriptase PCR revealed cardiac transcription factors and cardiac‐specific genes expression. CMs were electrically connected to one another. Recorded action potential (AP) showed waves of relatively mature ventricular‐like phenotype. Significance: We demonstrated that hiPSC lines respond differently to a standard cardiac differentiation protocol and that a well‐orchestrated interplay between Wnt, BMP4, FGF/MEK and Ascorbic acid MEK/ERK1/2 signaling pathways is beneficial in enhancing the differentiation outcome.

Detection of BCR/ABL Translocation in Bone Marrow Derived Mesenchymal Stem Cells in Egyptian CML Patients

BACKGROUND: Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder of hematopoietic stem cells. It is characterized at the cytogenetic level by Philadelphia (ph) chromosome and at the molecular level by the BCR/ABL gene rearrangement. Bone marrow derived mesenchymal stem cells (MSCs) are pluripotent stem cells that can differentiate into several mesenchymal tissues. AIM: To observe the biological characteristics of MSCS from CML patients and to determine whether MSCs harbor the abnormal BCR/ABL translocation similar to CML bone marrow cells. SUBJECTS AND METHODS: Characterized MSCs were isolated from 12 newly diagnosed Philadelphia positive untreated CML patients. RESULTS: MSCs can be readily isolated from CML marrow and exhibit major expansion. Flow cytometry analysis revealed the typical MSC phenotype. Moreover; MSCs do not harbor the BCR/ABL translocation confirmed by karyotype and real time PCR. CONCLUSION: MSCs from CML patients express the typical MSC phenotype; and do not express the BCR/ABL gene. Since; MSCs are able to support engraftment of hematopoietic stem cells in stem cell transplantation(SCT) as well as suppress alloreactive T cells causing graft versus –host disease, this current study provides evidence that in a SCT setting of CML patients, autologous MSCs could be a source of stem cell support in future cell therapy applications.