Heba Gouda

Ex vivo expansion of stem cells: defining optimum conditions using various cytokines.

With the increasing information on the number, quality, and characteristics of hematopoietic stem cells (HSC) in umbilical cord and placental blood, this material has been found to be efficacious as an alternative source of HSC for transplantation in children. In this study, we sought to define the optimal conditions for ex vivo expansion of cord blood (CB) stem cells. These conditions include: the combinations and concentrations of hematopoietic growth factors (stem cell factor [SCF], granulocyte-macrophage colony-stimulating factor [GM-CSF], interleukin [IL]-3, thrombopoietin [Tpo], IL-6 and Fms-like tyrosine kinase 3 ligand [Flt-3L]), the duration of culture, and the effect of serum supplementation. In this study, 2 protocols were applied for ex vivo expansion of CB stem cells. In protocol I, 20 CB samples were expanded in a static, serum-added, liquid culture for 7 and 11 days using 5 cytokine cocktails. In protocol II, 10 CB samples were expanded for 7 days using cytokines of cocktail 1, with and without IL-6 and Flt-3L, in serum-added and serum-free culture media. This protocol was intended to verify the effect of IL-6, Flt-3L, and the role of serum supplementation in short-term liquid culture. From the present study, it can be concluded that cocktail 1 is the cocktail of choice for ex vivo expansion of CB stem cells in serum-free, liquid culture expanded for 7 days. We can also conclude that culture expanded for 7 days is better than 11 days, as the fold expansion of CD34+ cells was not significantly increased or even decreased in some of the cocktails used. Moreover, the percent of CD95+ cells (apoptotic cells) was significantly increased on day 11 compared to day 7 in the cocktails tested.

Autologous melanocyte-keratinocyte suspension in the treatment of vitiligo.

Background  In stable vitiligo, several techniques of autologous transplantation of melanocytes are used. Autologous melanocyte transplantation of non‐cultured melanocytes is one of those techniques with variable reported outcomes.

The association between hepatitis C virus infection, genetic polymorphisms of oxidative stress genes and B-cell non-Hodgkin's lymphoma risk in Egypt.

Hepatitis C virus (HCV) has been postulated to be an etiological agent for lymphoid malignancies. Polymorphisms in oxidative stress genes as; superoxide dismutase (SOD2), glutathione peroxidase (GPX1), catalase (CAT), myeloperoxidase (MPO) and nitric oxide synthase (NOS2) may influence non-Hodgkins lymphoma (NHL) risk. HCV screening and polymorphisms in these five genes coding for antioxidant enzymes were studied in 100 Egyptian patients with B cell-NHL and 100 controls to clarify the association between HCV infection, oxidative stress genes polymorphisms and B cell-NHL risk. A significantly higher prevalence of HCV infection was detected among NHL patients relative to controls and this carried a 14-fold increased NHL risk (odds ratio (OR)=14.3, 95% confidence interval (CI)=5.4-38.3, p<0.0001). GPX1 and MPO genetic polymorphisms conveyed increase in B-NHL risk (OR=3.3, 95% CI=1.4-7.4, p=0.004 and OR=4.4, 95% CI=1.3-14.2, p=0.009 respectively). Further analyses stratified by HCV infection revealed that concomitant HCV infection and GPX1 gene polymorphism had a synergetic effect on NHL risk with an OR of 15 (95%CI=2.2-69.6, p<0.0001). In addition, combined HCV infection and MPO gene polymorphisms had a pronounced NHL risk (OR=9.2, 95%CI=2.5-33.9, p<0.0001). SOD2, CAT and NOS2 genetic polymorphisms were not found to confer increased NHL risk. This study revealed that HCV infection is a risk factor for NHL in Egypt. Polymorphisms in GPX1 and MPO genes may influence NHL risk in HCV infected Egyptian patients. Larger scale studies are warranted to establish this genetic susceptibility for NHL.

Association of cytotoxic T-lymphocyte antigen 4 genetic polymorphism, hepatitis C viral infection and B-cell non-Hodgkin lymphoma: an Egyptian study

Abstract Genetic and environmental factors are involved in the pathogenesis of non-Hodgkin lymphoma (NHL). The present study aimed to investigate the association between cytotoxic T-lymphocyte antigen 4 (CTLA-4) genetic polymorphism, hepatitis C virus (HCV) infection and B-cell NHL risk in Egypt. Genotyping of CTLA-4 single nucleotide polymorphisms (SNPs) was performed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay for 181 adult patients with B-NHL and 200 controls. Our study revealed that CTLA-4 + 49 A/G polymorphism conferred increased risk of B-NHL (odds ratio [OR] = 1.7, 95% confidence interval [CI] = 1.36–2.565). The prevalence of HCV infection in individuals harboring the mutant genotype + 49 A/G and − 318 C/T SNPs was higher in patients with B-NHL and was associated with increased risk of B-NHL (OR = 2.79, 95% CI = 1.24–6.93 for + 49 A/G and OR = 3.9, 95% CI = 1.01–15.98 for − 318 C/T). In conclusion, some SNPs of CTLA-4 are genetic risk factors for B-NHL. Moreover, this study identified an association of CTLA-4 + 49 A/G and − 318 C/T promoter polymorphisms with HCV infection.

Diagnostic values of CD64, C-reactive protein and procalcitonin in ventilator-associated pneumonia in adult trauma patients: a pilot study.

Abstract Background: Ventilator-associated pneumonia (VAP) is one of the most common nosocomial infections; however, its diagnosis remains difficult to establish in the critical care setting. We investigated the potential role of neutrophil CD64 (nCD64) expression as an early marker for the diagnosis of VAP. Methods: Forty-nine consecutive patients with clinically suspected VAP were prospectively included in a single-center study. The levels of nCD64, C-reactive protein (CRP), and serum procalcitonin (PCT) were analyzed for diagnostic evaluation at the time of intubation (baseline), at day 0 (time of diagnosis), and at day 3. The receiver operating characteristic curves were analyzed to identify the ideal cutoff values. Results: VAP was confirmed in 36 of 49 cases. In patients with and without VAP, the median levels (interquartile range, IQR) of nCD64 did not differ either at baseline [2.4 (IQR, 1.8–3.1) and 2.6 (IQR, 2.3–3.2), respectively; p=0.3] or at day 0 [2 (IQR, 2.5–3.0) and 2.6 (IQR, 2.4–2.9), respectively; p=0.8]. CRP showed the largest area under the curve (AUC) at day 3. The optimum cutoff value for CRP according to the maximum Youden index was 133 mg/dL. This cutoff value had 69% sensitivity and 76% specificity for predicting VAP; the AUC was 0.73 (95% CI, 0.59–0.85). The nCD64 and PCT values could not discriminate between the VAP and non-VAP groups either at day 0 or day 3. Conclusions: The results of this pilot study suggest that neutrophil CD64 measurement has a poor role in facilitating the diagnosis of VAP and thus may not be practically recommended to guide the administration of antibiotics when VAP is suspected.

Effect of Procedural-Related Variables on Melanocyte-Keratinocyte Suspension Transplantation in Nonsegmental Stable Vitiligo: A Clinical and Immunocytochemical Study.

BACKGROUND Melanocyte–keratinocyte suspension (M–K susp) is gaining popularity for vitiligo treatment. Few studies have addressed procedure-related variables. OBJECTIVE To assess the effect of different M–K susp procedure-related variables on the clinical outcome in stable vitiligo. METHODS This prospective multicenter comparative study included 40 cases with nonsegmental stable vitiligo. Donor site was either a skin graft in noncultured epidermal cell suspension (NCECS) or hair follicle units in outer root sheath hair follicle suspension (ORSHFS). Recipient site was prepared by either cryoblebbing or CO2 laser resurfacing. Cell counts and viability were recorded in the cell suspensions. Tissue melanocytes and keratinocytes were examined by melan-A and cytokeratin, respectively. Assessment of repigmentation was performed 18 months after the procedure. RESULTS Thirty-seven subjects completed the study. Cell count was significantly lower in the ORSHFS compared with NCECS with no significant difference in the repigmentation outcome. On comparing techniques of recipient site preparation, homogenicity was better in the CO2 group. Elbows and knees responded better to CO2 resurfacing, whereas distal fingers responded better to combination of cryoblebbing with NCECS. CONCLUSION Using different techniques in M–K susp produces comparable results. However, the distal fingers showed better results using combination of donor NCECS and recipient cryoblebs.

Differentiation of insulin-producing cells from human cord bloodderived haemopoietic stem cells in vitro

The use of stem cells in regenerative medicine holds great promise for the cure of many diseases, including type 1 diabetes mellitus, which, despite of the advances in current therapeutic approaches, remains to be one of the most serious health care problems. In addition to the traditional sources of adult stem cells, human umbilical cord blood has provided an important source of stem cells for research due to its unique advantages compared to other sources. In this study, we aimed at optimizing culture conditions for obtaining insulin-producing cells from cord blood hematopoietic stem cells. Twenty cord blood samples were subjected to short-term, liquid static culture that favors the proliferation of CD34+ hematopoietic stem cells. A duplicate culture was set for each sample, one with high glucose concentration and the other with low glucose concentration. Then these cells were subsequently induced to transdifferentiate into insulin-producing cells via a biphasic liquid culture using exendin-4. The expression of human insulin was then tested using RT-PCR. At the end of the culture, 17 out of the 20 samples (85%) cultured in high glucose concentration showed positive human insulin mRNA expression, while culture media with low glucose concentration failed to induce transdifferentiation into insulin-producing cells in any of the 20 samples. In brief, our study demonstrate that hematopoietic cord blood stem cells can transdifferentiate into insulin-producing cells in short-term liquid culture supplemented with high glucose concentration, nicotinamide, and exendin-4 in vitro.

The clinical significance of methylenetetrahydrofolate reductase (MTHFR) polymorphisms in acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Genetic polymorphisms in the folate metabolic pathway may contribute to the susceptibility to childhood ALL because they affect the DNA synthesis, methylation, and repair. The most common polymorphisms are methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C. The current study aimed at detecting the frequency of these two genetic polymorphisms in de novo ALL patients, and to clarify their impact on the response to induction chemotherapy, as well as treatment toxicity. MTHFR C677T and A1298C polymorphisms were tested in 30 de novo ALL patients by restriction fragment length polymerase chain reaction technique. Thirty normal age- and sex-matched subjects were subjected to the same analysis as a control group. The frequency of MTHFR A1298C gene polymorphism was significantly lower in ALL patients than the controls thus showing a protective effect. The two polymorphisms had no effect on the response to induction chemotherapy. As regards the treatment toxicity, MTHFR C677T polymorphism was associated with marked thrombocytopenia, while A1298C polymorphism was associated with hepatic toxicity. Identifying predictors of methotrexate sensitivity may lead to the development of individualized treatment strategies with improved efficacy and reduced toxicity as well as adjusting the initial methotrexate dose.

Clinical relevance of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 expression in acute myeloid leukemia

The angiogenic-related factors, angiopoietin-1 and angiopoietin-2 and their receptor Tie-2, have wide-ranging effects on tumor behavior that includes angiogenesis and inflammation. These multifaceted pathways present a potential target in developing novel inhibition strategies for cancer therapy. The present work aimed at detecting the prevalence of expression of angiopoietin-1, angiopoietin-2, and their receptor Tie-2 in 56 Egyptian de novo acute myeloid leukemia (AML) patients by conventional RT-PCR to verify the prognostic impact of their expression on the response to induction chemotherapy. Thirty age- and sex-matched healthy volunteers were subjected to the same analysis as a control group. High expression of Ang-1 was detected in the patient group but not the control group. AML patients expressing Ang-2 either solely or in combination with high Ang-1 and/or Tie-2 showed unfavorable response to induction chemotherapy, either failed induction or death during induction. These data provide evidence that the alternation of angiopoietin balance in favor of Ang-2 may play a critical role in the pathophysiology of AML. Furthermore, positive pre-therapeutic expression of Ang-2 indicates viable unfavorable prognostic marker in AML patients and may be used as a prognostic tool in the risk-adaptive management of AML.