Genetic polymorphisms effect on cyclophosphamide's tolerability and clinical efficacy in Egyptian patients with lupus nephritis.

OBJECTIVES: Many studies were conducted to determine the association between genetic polymorphisms in CYP2B6 c.516G>T and cyclophosphamide (CYC) efficacy or toxicity, no studies were focused on both clinical efficacy and toxicity of CYC. This study aimed to investigate the relationship between the CYP2B6 c.516G>T polymorphism (rs 3745274) and 17 different parameters related to CYC efficacy and tolerability in Egyptian patients with lupus nephritis (LN). METHODS: A prospective cohort study on 142 LN patients with a mean age of 36.26 was conducted at Kasr Al Ainy School of Medicine, Cairo University, Egypt after the exclusion of 14 patients due to receiving an interacting medication with CYC. All clinical parameters related to CYC efficacy or toxicity were recorded and compared between the different genotypes. RESULTS: There was a statistically significant difference between different genotypes in 11 out of 13 of the studied efficacy-related parameters. Many of the studied clinical parameters revealed that CYC's efficacy was associated with the presence of the T allele. There was a statistically significant difference between different genotypes in hepatotoxicity, diarrhea, and blood-related toxicities. CONCLUSION: To our knowledge, this study is the first study that focused on studying 17 different parameters related to CYC efficacy and tolerability. Our findings paint a picture of the function that CYP2B6 polymorphisms play in Egyptian LN patients. Pre-treatment evaluation of CYP2B6 rs 3745274 may account for some individual differences in treatment response.

Lab-scale production of anti-Schistosoma monoclonal antibodies by hybridoma culturing in serum free medium: Enhancement of growth, cryopreservation survival rate, and diagnostic accuracy.

This study was performed to evaluate the effect of two commercially available serum-free culture media; serum free medium (SFM) and chemically defined medium (CDM), on the growth rate, antibody productivity and post adaptation cryopreservation and revival reactivity of hybridoma cells compared to the conventional serum based medium (SBM). In addition, the diagnostic efficacy of MAbs secreted in each culture medium was evaluated by testing their performance in sandwich ELISA for antigen detection. Anti- Schistosoma mansoni soluble egg antigen hybridoma cell line (7A/8F) secreting previously characterized IgG Kappa mAbs, were retrieved and propagated in each of the three aforementioned media. Growth rate and viability were assessed post culturing in each media. The data collected from this study indicated that MAbs secreted from hybridoma cells cultured in SFM were the most abundant, easiest to purify, and the most effective in antigen detection by sandwich ELISA, in comparison to those produced in the other two media. Moreover, combination of fresh and conditioned medium with DMSO 7.5% was the most promising formulation for the cryopreservation of hybridoma cells cultivated in serum independent media (SFM or CDM).

Role of ApoB-516C/T promoter gene polymorphism in the risk of Hepatitis C virus infection in Egyptian patients and in gender susceptibility.

At least 1 in 10 of the Egyptian population aged 15-59 is burdened with hepatitis C virus (HCV) infection, stamping Egypt the highest country harboring HCV worldwide. Considerable evidence supported the involvement of host genetic factors in the pathogenesis of HCV and the possibility of implementation in target therapies. ApoB gene polymorphisms are postulated to affect the susceptibility of HCV infection. Hence, we aimed to evaluate the relationship between ApoB-516C/T promoter gene polymorphism and HCV infection in a cohort of Egyptian patients and to explore whether higher levels of low-density lipoprotein (LDL) might compete with lipoviral particles (LVP) in the binding to LDL receptor (LDLR), thus escaping infection. Ninety-seven HCV patients and 96 matched controls were enrolled in this study. We genotyped ApoB-516C/T using PCR-RFLP method. ApoB concentrations were measured by immunoturbidimetric assay. The genotype and the allele frequencies of ApoB-516C/T promoter gene polymorphism in cases were statistically insignificant compared with healthy individuals (P = 0.109, 0.125, respectively). Sex stratification showed significantly lower counts of C/T genotype in female patients compared with female controls (P = 0.011, OR = 0.132, 95% CI = 0.026-0.657). Significantly higher levels of LDL and ApoB were detected in the control group (P < 0.001). This study shows that the ApoB-516C/T promoter gene polymorphism has no impact on the risk of HCV infection. However, the C/T genotype may be a protective factor for our female cohort. Further studies with larger samples are needed to verify this genetic gender diversity. Additionally, high levels of LDL and ApoB might prevent HCV infection.

Contribution of Toll-Like Receptor 9 Gene Single-Nucleotide Polymorphism to Systemic Lupus Erythematosus in Egyptian Patients.

BACKGROUND: Systemic lupus erythematosus (SLE) is an autoimmune disease, with multiple genetic and environmental factors involved in its etiology. The toll-like receptor 9 (TLR9) gene has been reported to have important roles in the development and progression of SLE. In this case-control study, the effect of TLR9 polymorphism on susceptibility to SLE was investigated in Egyptian patients. METHODS: We studied the distribution of the TLR9 rs352139 (G + 1174A) single nucleotide polymorphism (SNP) by allele-specific polymerase chain reaction (PCR) in 104 Egyptian patients with SLE and 108 age-, sex-, and ethnically matched controls. RESULTS: There was no statistically significant difference in the distribution of the AA genotype and alleles between SLE patients and the control group in our study; however, the GA heterozygous patients were three times more likely to develop SLE (P < 0.001). A significant association was detected between TLR9 genotypes and some of the disease manifestations as myositis (p = 0.032), psychosis (p = 0.014), photosensitivity (p = 0.002), and pleurisy (p = <0.001). Moreover, we observed a significant association between the TLR9 AA and GA genotypes and the presence of antinuclear antibodies (ANA) (p = 0.038). CONCLUSION: The G + 1174A SNP in the toll receptor 9 gene may contribute to the genetic susceptibility of SLE in Egyptian patients. Also, an influence for this polymorphism on disease manifestations has been elucidated.

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.

Serial changes in renal indices in chronic HCV patients with and without HIV co-infection receiving sofosbuvir and tenofovir-based therapies.

BACKGROUND: Sofosbuvir (SOF) is authorized for hepatitis C virus (HCV) patients. The nephrotoxicity of SOF on HCV mono-infected and HCV-human immunodeficiency virus (HIV) individuals receiving antiretroviral therapy (ART) remains controversial. METHODS: A prospective study including 159 HCV mono-infected and 124 HCV-HIV individuals (47 were ART naïve and 77 were tenofovir [TDF]-based ART) who presented with an estimated glomerular filtration rate (eGFR) ≥30 ml/min/1.73 m2 at baseline and were treated with SOF-daclatasvir for 12 weeks. The eGFR was estimated using the Chronic Kidney Disease Epidemiology Collaboration equation over the study period. RESULTS: HCV patients had a progressive decline in median levels of eGFR compared with HCV-HIV patients who were ART naïve and those receiving TDF-based ART during and after discontinuing SOF-DAC treatment (96, 109 and 114 at baseline vs 94, 117 and 108 at the end of treatment [EOT]) vs 95, 114 and 115 ml/min/1.73 m2 at 12 weeks after treatment [SVR12], respectively). Moreover, the rate of eGFR stage worsening was more pronounced in HCV mono-infected compared with HCV-HIV individuals who were ART naïve and those receiving TDF-based ART (21.4% vs 8.5% and 14.3% at EOT; 21.4% vs 2.1% and 6.5% at SVR12, respectively). Multivariable regression analysis showed that baseline variables were not independent predictors of eGFR stage worsening either at EOT or SVR12. CONCLUSIONS: Because the changes in eGFR were minimal and not of clinical significance, and TDF was not associated with an increase in renal dysfunction, SOF-based direct-acting antivirals could be safely used in HCV mono-infected and HCV-HIV individuals, even in those on TDF-based ART.

Evaluating sex hormones and cytokine profile in Egyptian females with relapsing-remitting multiple sclerosis.

BACKGROUND: Sexual dimorphism shown in multiple sclerosis suggests an interaction between immune system and sex hormones. The objective of this study is to determine the hormonal profile and serum cytokine levels in Egyptian female patients with relapsing-remitting MS (RRMS) compared with healthy controls and their associations with disease disability. METHODS: This study was conducted on 40 female patients with RRMS and 20 age-matched controls subjected to measurements of the hormonal profile (estrogen, testosterone) and cytokine levels (interleukin 10 and 4 and tumor necrosis factor alpha) and disability assessment using Expanded Disability Status Scale (EDSS). RESULTS: Levels of estrogen, testosterone, interleukin 10 and 4 (IL-10 and IL-4), and tumor necrosis factor alpha (TNF-α) were higher in patients compared to control with no statistically significant difference. Estrogen levels were positively correlated with interleukin 10 and interleukin 4 levels and negatively correlated with tumor necrosis factor alpha (TNF-α), but there was no statistically significant correlation between hormonal profile or cytokine profile (IL-10, IL-4, and TNF-α) and EDSS. CONCLUSIONS: It is suggested that estrogen has an anti-inflammatory effect on cytokine milieu; therefore, it can be tried as a treatment option in multiple sclerosis.

The Effect of Monophosphoryl Lipid A on Maturation of DCs from Patients with Acute Myeloid Leukaemia.

BACKGROUND: Generation of monocyte-derived dendritic cells (MDDC) is induced in the presence of GM-CSF and IL-4, and a maturation stimulus is added to the monocyte culture to obtain mature Dendritic Cells (DCs) suitable for therapy. TNF-α is the most common cytokine used for activating DCs and generating mature MDDC either alone or in combination with other cytokines. OBJECTIVE: To compare effects of traditional cytokine cocktail (TNF-α + IL-1ß) versus TLR4-agonist monophosphoryl lipid A on the viability, phenotype, cytokine profile and functionality of MDDC. METHODS: The study included 32 individuals; twenty Acute Myeloid Leukaemia (AML) cases in complete remission and 12 healthy volunteers. They were divided into 3 groups: Group 1: control group: 12 subjects to measure the baseline levels of all markers in the monocytic preparation. Group 2: cytokine cocktail (TNF-α) group, which included 10 AML subjects. Group 3: MPLA group which included 10 AML subjects. RESULTS: TNF-α group showed higher expression of CD83 than MPLA group indicating higher capacity to induce DC maturation but both were similar in CD86, CCR7 and IL-10 expression. Preparation of dendritic cells from AML cases in remission and loading them with tumor peptides was successful. CONCLUSION: MPLA effect in DC maturation is comparable with traditional DC maturation cocktail.