Diagnosis of DOCK8 deficiency using Flow cytometry Biomarkers: an Egyptian Center experience., Meshaal, Safa S., El Hawary Rabab E., Eldash Alia, Grimbacher Bodo, Camacho-Ordonez Nadezhda, Abd Elaziz Dalia S., Galal Nermeen M., Boutros Jeannette A., Shawky Shereen M., and Elmarsafy Aisha M. , Clinical immunology (Orlando, Fla.), 2018 Oct, Volume 195, p.36-44, (2018) Abstract

In the past few years, several genes were shown to be implicated in various forms of the Hyper Immunoglobulin E syndrome. The present study is the first to describe a cohort of DOCK8 deficiency patients from Egypt. The study included 15 patients with features of combined immunodeficiency (CID) suggestive of DOCK8 deficiency. Flow cytometry was used for evaluation of DOCK8 expression and studying different immunological characteristics of those patients including evaluation of Th17, Tregs, T and B lymphocytes differentiation and the effect of the DOCK8 deficiency on the activation of the STAT3. Diagnosis was confirmed by mutational analysis. Profound defects in Th17 cells and Tregs were observed in all patients with impaired STAT3 phosphorylation, indicating that DOCK8 plays a pivotal role in the STAT3 signaling pathway. These findings together with decrease in memory B cells and defective DOCK8 expression by flow cytometry can confirm the diagnosis.

Utilizing the KCNJ11 Gene Mutations in Spotting Egyptian Patients With Permanent Neonatal Diabetes Who Can Benefit From Treatment Shift, Ahmed, Dina M., Dayem Soha Abd El M., Kader Mona Abdel, Khalifa Rania H., El-Lebedy Dalia H., Kamel Solaf A., and Shawky Shereen M. , Laboratory Medicine, Volume 48, Issue 31, p.225–229, (2017) AbstractWebsite

Background
Neonatal diabetes mellitus (NDM) is a monogenic form of diabetes mellitus. Until now, patients in developing countries who had this condition had been misdiagnosed as having type 1 diabetes mellitus and accordingly directed to erroneous, ineffective, and costly therapeutic regimens.

Objective
To detect Egyptian patients who harbor pathological variant in the KCNJ11 gene, so that their treatment regimen can be modified as needed to increase its effectiveness.

Methods
We sequenced KCNJ11 in 17 ethnic Egyptian probands diagnosed with diabetes mellitus before age 2 years.

Results
A preliminary case individual harboring a KCNJ11 pathological variant (p.R201H) was identified. The patient was successfully shifted from insulin therapy to sulfonylurea. Four previously identified benign variants, namely, E23K, I337V, L270V, and A190A, were detected in this patient.

Conclusion
Implementing the findings of this molecular analysis could have a major clinical and nationwide economic impact on world health, especially in developing countries.

Long term culture of CB-MSCs deteriorates their stemness nature through a series of biomolecular changes, Maher, Kesmat, El-Baz Hanan, Demerdash Zeinab, Shawky Shereen Mahmoud, Hassan Marwa, Mohamed Salwa, Mahmoud Faten, Hammam Olfat, and Taha Tamer , Global Journal of Medicine and Medical Sciences, Volume 3, Issue 1, p.097-107, (2015) Abstract

This study was designed to evaluate the effect of long term in vitro culture on morphology, proliferation, telomere length, pluripotency genes expression, and differentiation potentials of cord blood (CB)-derived MSCs. This would allow us to choose the optimum passage number maintaining the MSCs stemness nature and providing the sufficient count intended for their clinical applications. Upon repeated passaging, CB-MSCs telomere length has decreased from 10.0168 kb ± 0.27 to 7.4186 kb ± 0.996 and this decrease was statistically highly significant (p < 0.01) which coincided with negative telomerase activity. Pluripotency and proliferation genes expression also decreased at the late passages. The adipogenic and chondrogenic differentiation potentials declined gradually whereas the osteogenic differentiation increased gradually and then dropped at the late passages. Based on the results, in vitro expansion attenuated the parameters that were commonly used to define CB-MSCs stemness nature except for the osteogenic differentiation potential which was highest at the intermediate passages. Therefore, we suggest considering CB-MSCs for cell and gene therapy at an early passage, as soon as a clinically sufficient MSCs count is achieved. For orthopaedic applications, the intermediate passages of CB-MSCs will be the best.

Immunomodulatory Effect Of Umbilical Cord derived Mesenchymal Stem Cells., Shawki, Shereen, Gaafar Taghrid, Erfan Hadeel, Khateeb Engy El, Sheikhah Ahmad El, and Hawary Rabab El , Microbiology and immunology, 2015 Apr 13, (2015) Abstract

Umbilical cord blood (UCB) gained high interest as a source of stem cells for use in cellular therapies. The immune modulatory effect of mesenchymal stem cells (MSCs) originating from bone marrow, adipose tissue and amniotic membrane was previously proved. In this study we aimed at isolation of MSCs from UCB, and evaluate their immune-modulatory effect on peripheral blood lymphocytes proliferation by two different techniques; 5-Bromo-2-deoxyuridine (Brdu) enzyme linked immunsorbant assay (ELISA) and Carboxy Fluorescine diacetate Succinimidyl Ester (CFSE) flow cytometric technique. MSCs were isolated from UCB, propagated until passage 4, characterized for cell surface markers by flow cytometry and ability of mesodermal differentiation was induced towards osteocytes and adipocytes. The immunosuppressive effect on peripheral blood lymphocytes was examined by co-culturing Mitomycin C treated UCB MSCs with mitogen-stimulated lymphocytes for 72 hours. Thereafter, proliferation of lymphocytes was detected by CFSE flow cytometry and colorimetric ELISA. The level of cytokines in cell culture supernatant was also assayed to detect the possible mechanism of immunomodulation. UCB MSCs were able to suppress mitogen-stimulated lymphocytes proliferation; this is due to cell-cell contact as well as due to cytokines secretion. The level of transforming growth factor beta (TGFβ) and interleukin (IL) 10 increased and the level of interferon gamma (IFNγ) decreased in the supernatant of co-cultures. UCB MSCs suppressed the proliferation of mitogen-stimulated lymphocytes. However further in vivo studies are still required to fully evaluate the immune-modulatory effect of UCB MSCs.

Increased DNA Damage in Hepatitis C Virus-Related Hepatocellular Carcinoma, Shawki, Shereen, Meshaal Safa S., Dash Aliaa El S., Zayed Naglaa, and Hanna Mariam Onsy F. , Journal of Hepatology , Volume 33, Issue 12, p.884-890, (2014) Abstract

One consequence of hepatitis C virus (HCV) infection is an elevated cancer risk. During chronic viral infection, deoxyribonucleic acid (DNA) damage is being induced by reactive oxygen and nitrogen species, which may play a pathogenic role in HCV-induced carcinogenesis. The study investigated DNA damage in peripheral blood lymphocytes from patients with hepatocellular carcinoma (HCC) and those with HCV infection with and without associated cirrhosis and normal controls. As a measure for genomic damage, the comet assay (single cell gel electrophoresis) was applied, which detects single- and double-strand breaks and alkali-labile sites through electrophoretic mobility of the resulting fragments. The levels of DNA damage were significantly higher in HCC and HCV-associated cirrhosis compared to HCV without cirrhosis and the control group. Patients presenting with DNA damage more than mean+two standard deviation of the controls had a 3.6-fold risk of having HCC more than those with undamaged DNA. HCV disease progression was the only discriminator predicting the extent of DNA damage. The accumulation of DNA damage is important in HCC evolution. DNA damage indicating intracellular oxidative and nitrative stress may lead to mutagenesis and consequently malignant transformation, which emphasizes the need to optimize the therapy for reducing the degree of genomic damage.

Dendritic Cell Vaccine as an Adjuvant Therapy in the Treatment of Chronic Myeloid Leukemia, El-Ansary, Mervat, Mattar Mervat, Kamel Shereen, El-Hamid Samah Abd, and Elnour Rania Abou , Global Science of Books Dynamic Biochemistry , Process Biotechnology and molecular biology, Volume 4, Issue Special Issue, p.61-66, (2010)
Enhancing ex vivo expansion of cord blood-derived unrestricted somatic stem cells for clinical applications, Demerdash, Z., El Baz H., Mahmoud F., Mohamed S., Maher K., Gaafar T., Shawky S., Hassan M., Abdelhady D., and Taha T. , Cell Proliferation, Volume 46, Number 6, p.628–636, (2013) AbstractWebsite

Objectives To study effects of serum-containing medium (SCM) versus serum-free medium (SFM) and influence of seeding density, on rate of expansion of cord blood (CB) unrestricted somatic stem cells (USSCs), as a prerequisite for evaluating their therapeutic potential in ongoing clinical trials. Material and methods Isolation, propagation and characterization of USSCs from CB samples were performed and followed by their passage 3 culture in SCM and SFM, at cell densities of 5, 50, 500 and 5000 cells/cm2. Results The cells were CD44+, CD90+, CD73+, CD105+, CD34−, CD45−, and HLA-DR, with Oct4 & Sox2 gene expression; they were differentiated into osteoblasts and adipocytes. USSCs cultured in SCM had significantly higher population doubling levels (P < 0.01) than those cultured in SFM. Those cultured in SCM at 5 cells/cm2 and those cultured in SFM at 50 cells/cm2 had significantly higher population doubling (P < 0.01) levels than those cultured at higher cell densities. Conclusions For scaling up of USSCs from 106 (?) to 1012 (?) in 6 weeks, culturing of CB-derived cells of early passage (≤P3) in SCM at low cell seeding density (5 cells/cm2) is suggested for increasing cell count with lower passaging frequency, followed by culture of expanded USSCs at 50 cells/cm2 in SFM, to avoid undesirable effects of bovine serum in clinical applications.

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