Rabab El Hawary

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.

Signal transducer and activator of transcription 5 is implicated in disease activity in adult and juvenile onset systemic lupus erythematosus

The Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway is one of a handful of pleiotropic cascades used to transduce a multitude of signals for development and homeostasis in humans. It is the principal signaling mechanism for a wide array of cytokines and growth factors. Dysregulated cytokine action on immune cells plays an important role in the initiation and progress of systemic lupus erythematosus (SLE). In this study, we tried to assess the role of STAT5 in systemic lupus erythematosus and correlate its phosphorylation level with the disease activity. The activation of the STAT5 was assessed by measuring the level of expression of phosphorylated STAT5 (pSTAT5) using flow cytometry on the peripheral blood T and B cells in 58 SLE patients (40 adult and 18 juvenile onset) and on 23 healthy age- and sex-matched controls for both groups. Serum prolactin level was also assessed in the patients and control by ELISA. The study revealed that the level of pSTAT5 was higher in adult SLE patients than in healthy control (p = 0.001) and in juvenile-onset SLE patients versus age-matched control (p = 0.031). A positive correlation existed between the pSTAT5 levels and Systemic Lupus Activity Measure (SLAM) score and also with multiple clinical manifestations indicating a potential role of STAT5 signaling in pathogenesis SLE. The pSTAT5 signaling is implicated in the disease activity of SLE and may be a useful target of therapy by correcting the dysregulation of cytokines involved in the disease pathogenesis.

Mutations in Recombination Activating Gene 1 and 2 in patients with severe combined immunodeficiency disorders in Egypt

The Recombination Activating Genes (RAG) 1/2 are important for the development and function of T and B cells. Loss of RAG1/2 function results in severe combined immunodeficiency (SCID), which could lead to early death. We studied the prevalence of RAG1/2 mutations in ten SCID patients in Egypt. We identified two novel homozygous nonsense mutations in RAG1, a novel homozygous deletion, and a previously reported homozygous missense mutation from four patients, as well as two homozygous mutations in RAG2 from the same patient. Prenatal diagnosis performed in the mother of a patient with RAG1 deficiency determined that the fetus was heterozygous for the same mutation. This represents the first report on RAG1/2 mutations in SCID patients in Egypt. The early diagnosis dramatically affects the outcome of the disease by allowing bone marrow transplantation at an early age, and providing prenatal diagnosis and genetic counseling for families with a history of SCID.

Phenotypic Heterogeneity in RAG-deficient Patients from a Highly Consanguineous Population

Mutations affecting recombination activation genes RAG1 and RAG2 are associated with variable phenotypes, depending on the residual recombinase activity. The aim of this study is to describe a variety of clinical phenotypes in RAG‐deficient patients from the highly consanguineous Egyptian population. Thirty‐one patients with RAG mutations (from 28 families) were included from 2013 to 2017. On the basis of clinical, immunological and genetic data, patients were subdivided into three groups; classical T–B– severe combined immunodeficiency (SCID), Omenn syndrome (OS) and atypical SCID. Nineteen patients presented with typical T–B–SCID; among these, five patients carried a homozygous RAG2 mutation G35V and five others carried two homozygous RAG2 mutations (T215I and R229Q) that were detected together. Four novel mutations were reported in the T–B–SCID group; three in RAG1 (A565P, N591Pfs*14 and K621E) and one in RAG2 (F29S). Seven patients presented with OS and a novel RAG2 mutation (C419W) was documented in one patient. The atypical SCID group comprised five patients. Two had normal B cell counts; one had a previously undescribed RAG2 mutation (V327D). The other three patients presented with autoimmune cytopaenias and features of combined immunodeficiency and were diagnosed at a relatively late age and with a substantial diagnostic delay; one patient had a novel RAG1 mutation (C335R). PID disorders are frequent among Egyptian children because of the high consanguinity. RAG mutations stand behind several variable phenotypes, including classical SCID, OS, atypical SCID with autoimmunity and T–B+ CID.

Diagnosis of DOCK8 deficiency using Flow cytometry Biomarkers: an Egyptian Center experience

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.

Genetic Counseling in Primary Immunodeficiency Disorders: An Emerging Experience in Egypt

BackgroundPrimary immunodeficiency disorders (PIDs) are a heterogeneous group of diseases of the immune system leading to life-threatening infections, and, unless urgently treated with immune reconstitution, patients do not usually survive. With the continuing progress in molecular diagnosis, many mutations have been described in more than 300 genes. Genetic counseling has recently been considered an essential part of the management of PIDs. This study presents the experience of genetic counseling services in the largest PID center in Egypt, and reports on our management plan and the impact of prenatal diagnosis (PND) on families.MethodsBased on the biochemical and molecular diagnosis of index cases, PND was offered for 10 families in 12 subsequent pregnancies. Five different genes were sequenced by Sanger sequencing in fetal samples.ResultsSeven fetuses were either normal or were carriers, while five fetuses were affected and human leukocyte antigen typing was performed, seeking a suitably related donor for stem cell transplantation.ConclusionIn spite of the genetic heterogeneity behind PIDs, genetic counseling should play a critical role in the management and future decisions of affected families.

Mediterranean fever gene mutations: correlation with cytotoxic T-lymphocyte-associated antigen 4 gene polymorphism.

Mutations in the Mediterranean fever (MEFV) gene lead to familial Mediterranean fever (FMF), a pro‐inflammatory state characterized by outbursts of inflammatory cytokines. The aims of this study were to identify the common mutations of MEFV gene in Egyptian patients with FMF, to study cytotoxic T lymphocyte associated antigen 4 (CTLA‐4) gene polymorphism and to evaluate correlations between CTLA4–1661 polymorphisms and MEFV mutations and clinical symptoms. Four hundred and twenty‐four patients with clinical pictures suspicious of FMF were enrolled in this study. Mutations in MEFV gene were confirmed by reversed hybridization. Patients with homozygous and compound heterozygous mutations and 120 healthy controls were investigated for polymorphism of −1661 CTLA4 gene and the findings correlated with disease incidence and clinical symptoms of the disease. Ninety‐seven patients had single heterozygous mutations and 78 had compound heterozygous or homozygous MEFV gene mutations. M694I/V726A was the most common genotype (14.1%), followed by homozygous M694I. There was no statistically significant difference between patients and controls in incidence of −1661 A/G single nucleotide polymorphism CTLA4 (P = 0.189), nor any significant correlation with any of the clinical symptoms of FMF and MEFV gene mutations.

Study of naïve and memory cells in a cohort of Egyptian chronic granulomatous disease patients

Abstract Context: Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder caused by inherited defects in the NADPH oxidase complex which may be involved in important pathways that connect innate and adaptive immunity. Objectives: Characterize the naive and memory compartment of B and T lymphocytes in patients with CGD. Methods: Twenty CGD patients and twenty healthy controls matched for age and sex were enrolled in this study. Flow cytometric assessment of the naïve and memory compartments of peripheral blood lymphocytes was done using cell surface markers CD45RO, CD45RA, CD27, CD3 and CD19. Results: There were 15 (79%) autosomal recessive CGD patients (8 females (53%) and 7 males (47%), 100% positive parental consanguinity) and four (21%) X-linked CGD patients. On comparing the 3 groups; AR CGD, X-linked CGD and controls, there was a positive statistical significant difference for the percentage and absolute count of CD19 + CD27+ memory B cell (p = 0.028 and p = 0.047 respectively), CD45RA cells (with p values of p = 0.000 and 0.033, respectively), the naïve compartment CD3 + CD45RA+ cells percentage and absolute counts (p = 0.005, 0.01respectively), CD3 + CD27 + cells percentage and absolute counts (p = 0.001, 0.012 respectively), CD3 + CD45RA + CD27+ cells percentage and absolute counts (p = 0.015, 0.005, respectively). The significance was mainly attributed to the decrease in the X-linked group than control group. Conclusion: There was an altered naïve and memory B profile in CGD patients, this may increase susceptibility of the patients to opportunistic infections and autoimmune disorders. T-cell alterations have to be interpreted cautiously especially in the presence of infections.