SARS-CoV-2 antibody seroprevalence rates among Egyptian blood donors around the third wave: Cross-sectional study.

Background: Seroprevalence studies may provide a more representative situation of the disease burden and population-level immunity in a country. Aim: The aim of this study was to determine the prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies among asymptomatic blood donors attending the Cairo University blood bank services at various points in time around the third wave. Methods: This cross-section study included 3058 eligible blood donors, representing a demographically and socially heterogeneous healthy population and categorized as: Group 1, 954 donors in the period from March 20 to 30/2021; Group 2, 990 donors in the period from June 3 to 10/2021. These two groups were tested for IgG against SARS-CoV-2 nucleocapsid antigen (NC) to detect qualitative reactivity. Group 3, 1114 donors in the period from July 20 to 30/2021 were tested by the SARS-CoV-2 IgG II Quant assay for the quantitative detection of IgG antibodies, including neutralizing antibodies (antispike antibodies). Results: Donors' age ranged between 18 and 59 (mean 33.9 ± 9) years. There was no significant correlation between seroprevalence and gender, area of residence, ABO or Rh blood types, and occupation or education. Antibody prevalence was found to be 13.2% in Group 1, 19.2% in Group 2 (overall 16.2%), and 66% in Group 3. There were only 49 included cases vaccinated against COVID-19. Conclusion: We concluded that the significant increasing trend in seroprevalence rates during the third wave, March, June, and July, in Egypt, reflects a high cumulative incidence of seroconversion that mirrored the epidemic curve in its rise, fall, and nadir.

Induction of Hepatic Regeneration in an Experimental Model Using Hepatocyte-Differentiated Mesenchymal Stem Cells.

Mesenchymal stem cell (MSC)-based liver tissue engineering on nanofibrous scaffold holds great promise for cell-based therapy in liver injuries and end-stage liver failure treatments. MSCs were generated from umbilical cord blood. Hepatogenic differentiation was induced on two-dimensional (2D) and three-dimensional (3D) culture system and characterized by morphology, scanning electron microscopy, immunocytochemistry, and gene expression. Albumin and α-1 antitrypsin (AAT) in culture supernatants were measured. Differentiated cells were administered intravenous into a murine model of carbon tetra induced liver cirrhosis. After 12 weeks of injection, liver pathology was examined. The hepatogenic differentiated MSCs stained positively for albumin, alpha fetoprotein, HepPar1, cytokeratin 7 and 18, and OV6 with more mature cells, hexagonal in shape with central nuclei forming large sheets in groups in 3D culture system. AAT secretion and indocyanine green uptake were significantly increased in 3D system. In experimental model, MSC-3D treated group exhibited maximal restoration of liver architecture with absent septal fibrosis and marked improvement of alanine transaminase (ALT) and aspartate transaminase (AST), and mild increase in albumin. Both 3D and 2D culture system are effective in functional hepatogenic differentiation from MSCs and serve as a vehicle in liver tissue engineering. In vivo hepatogenic differentiation is more effective on 3D scaffold, with better functional recovery.

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

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 α-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.

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.

Role of Flow Cytometry in the Diagnosis of Chronic Granulomatous Disease: the Egyptian Experience.

INTRODUCTION: Chronic granulomatous disease (CGD) is an inherited mutational defect in any of the NADPH oxidase complex, CYBB (gp91-phox), NCF1 (p47-phox), CYBA (p22-phox), NCF2 (p67-phox), or NCF4 (p40-phox) leading to inability of phagocytes to perform effective respiratory burst and thus diminished killing of bacteria and fungi. The identification of defective proteins aids in establishing a diagnosis prior to genetic analysis, which is rather labor-intensive, expensive, and time-consuming. AIM: The present study aims at assessing the NADPH proteins by performing the intracellular staining with specific monoclonal antibodies and their assessment on flow cytometry. The use of flow cytometry is less laborious and faster to perform than western blot. It also confirms the diagnosis of CGD and detects the affected components allowing proper management of patients. MATERIALS AND METHODS: Twenty-eight patients from 25 different kindred, clinically suspected as CGD were recruited in Egypt. Dihydrorhodamine test was performed to confirm the diagnosis of the patients. Intracellular staining of NADPH components using specific monoclonal antibodies was performed followed by flow cytometric analysis. RESULTS: The present study revealed that the most common defective protein in our cohort is p22-phox, found in 13 patients (46.4 % of cases) followed by p47-phox in 8 patients (28.6 %), gp91-phox in 5 patients (17.9 %), and finally p67-phox in 2 patients (7.1 %). CONCLUSION: In countries with limited resources and yet large number of CGD patients, the analysis of the defective proteins by flow cytometry is an optimum solution for confirming the diagnosis and is a step for targeted sequencing in families seeking prenatal diagnosis.

Focused molecular analysis of small cell lung cancer: feasibility in routine clinical practice.

BACKGROUND: There is an urgent need to identify molecular signatures in small cell lung cancer (SCLC) that may select patients who are likely to respond to molecularly targeted therapies. In this study, we investigate the feasibility of undertaking focused molecular analyses on routine diagnostic biopsies in patients with SCLC. METHODS: A series of histopathologically confirmed formalin-fixed, paraffin-embedded SCLC specimens were analysed for epidermal growth factor receptors (EGFR), KRAS, NRAS and BRAF mutations, ALK gene rearrangements and MET amplification. EGFR and KRAS mutation testing was evaluated using real time polymerase chain reaction (RT-PCR cobas(®)), BRAF and NRAS mutations using multiplex PCR and capillary electrophoresis-single strand conformation analysis, and ALK and MET aberrations with fluorescent in situ hybridization. All genetic aberrations detected were validated independently. RESULTS: A total of 105 patients diagnosed with SCLC between July 1990 and September 2006 were included. 60 (57 %) patients had suitable tumour tissue for molecular testing. 25 patients were successfully evaluated for all six pre-defined molecular aberrations. Eleven patients failed all molecular analysis. No mutations in EGFR, KRAS and NRAS were detected, and no ALK gene rearrangements or MET gene amplifications were identified. A V600E substitution in BRAF was detected in a Caucasian male smoker diagnosed with SCLC with squamoid and glandular features. CONCLUSION: The paucity of patients with sufficient tumour tissue, quality of DNA extracted and low frequency of aberrations detected indicate that alternative molecular characterisation approaches are necessary, such as the use of circulating plasma DNA in patients with SCLC.

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.

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.

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.

Gene expression profiling of endometrium versus bone marrow-derived mesenchymal stem cells: upregulation of cytokine genes.

Postulated Stem/progenitor cells involved in endometrium regeneration are epithelial, mesenchymal, and endothelial. Bone marrow (BM) has been implicated in endometrial stem cells. We aimed at studying gene expression profiling of endometrial mesenchymal stem cells compared to BM MSCS to better understand their nature and functional phenotype. Endometrial tissues were obtained from premenopausal hysterectomies (n = 3), minced and enzymatically digested as well as Normal BM aspirates (n=3). Immunophenotyping, differentiation to mesoderm, and proliferation were studied. The expression profile of 84 genes relevant to mesenchymal stem cells was performed. Fold change calculations were determined with SA Biosciences data analysis software. VEGF, G-CSF, and GM-CSF in cultures supernatants of MSCs were assayed by Luminex immunoassay. Endo MSCs possess properties similar to BM MSCs. Cumulative population doubling was significantly higher in Endo MSCs compared to BM MSCs (p < 0.001). 52 core genes were shared between both generated MSCs including stemness, self-renewal, members of the Notch, TGFB, FGF, and WNT.16 downregulated genes (VCAM, IGF1)and 16 upregulated in Endo MSCs compared to BM (p < 0.05 â†’ fourfolds). They included mostly cytokine and growth factor genes G-CSF, GM-CSF, VWF, IL1b, GDF15, and KDR. VEGF and G-CSF levels were higher in Endo MSCs supernatants (p < 0.0001). Cells sharing MSC and endothelial cell characteristics could be isolated from the human endometrium. Endo MSCs share a core genetic profile with BM MSCs including stemness. They show upregulation of genes involved in vasculogenesis, angiogenesis, cell adhesion, growth proliferation, migration, and differentiation of endothelial cells, all contributing to endometrial function.

Comparative characteristics of endothelial-like cells derived from human adipose mesenchymal stem cells and umbilical cord blood-derived endothelial cells.

Adult peripheral blood contains a limited number of endothelial progenitor cells that can be isolated for treatment of ischemic diseases. The adipose tissue became an interesting source of stem cells for regenerative medicine. This study aimed to investigate the phenotype of cells obtained by culturing adipose-derived mesenchymal stem cells (ad-MSCs) in the presence of endothelial growth supplements compared to endothelial cells obtained from umbilical cord blood (UCB). Passage 3 ad-MSCs and mononuclear layer from UCB were cultured in presence of endothelial growth media for 3 weeks followed by their characterization by flow cytometry and polymerase chain reaction. After culture in endothelial inductive media, ad-MSCs expressed endothelial genes and some endothelial marker proteins as CD31 and CD34, respectively. Adipose tissue could be a reliable source for easy obtaining, expanding and differentiating MSCs into endothelial-like cells for autologous cell-based therapy.