EFFECT OF ANTI-TSLPR MONOCLONAL ANTIBODY ON VIABILITY, PROAPOPTOTIC GENES EXPRESSION, AND PRODUCTION OF PRO-INFLAMMATORY CYTOKINES IN MCF-7 AND A549 CELLS.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) are expressed in various cancer cells. However, their role in cancer development is not well defined. AIM: To investigate the effects of anti-TSLPR antibody on the viability, proapoptotic genes expression, and production of pro-inflammatory cytokines in MCF-7 and A549 cancer cells. MATERIALS AND METHODS: MCF-7 and A549 cells were exposed to anti-TSLPR monoclonal antibody for 24, 48, and 72 h. The effect on cell viability was examined by MTT assay. The expression levels of TP53, BAX, and CASP3 genes were evaluated by the quantitative reverse transcription polymerase chain reaction (qRT-PCR). Levels of interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and transforming growth factor (TGF-ß1) were measured by the enzyme-linked immunosorbent assay (ELISA). RESULTS: The treatment of MCF-7 cells with anti- TSLPR antibody slightly stimulates cell proliferation after 48 h and 72 h following initial cytotoxicity in 24 h with a significant reduction in IL-6 and TNF-α production. A significant increase in the BAX expression in anti-TSLPR treated cells at a concentration of 2.5 µg/ml at 24-h point was evident. In anti-TSLPR-treated A549 cells, no decrease in cell count was observed, and slight dose-dependent stimulation of cell proliferation was evident in 48 h and 72 h of culture. A significant increase in TP53, BAX, and CASP3 expression upon treatment with 2.5 µg/ml of anti-TSLPR was evident in A549 cells. CONCLUSION: The effects of anti-TSLPR on cell viability, proapoptotic gene expression, and production of pro-inflammatory cytokines (IL-6 and TNF-α) vary in MCF-7 and A549 cells.

Bone morphogenic protein-7 (BMP-7) polymorphism: Susceptibility to cirrhosis and hepatocellular carcinoma after viral hepatitis in Egyptian patients.

Aim of the study: Bone morphogenic proteins (BMPs) have both inhibitory and stimulatory effects on growth of a tumor that depend on the type of cells, the dosage and the tumor microenvironment. We aimed to investigate the impact of the bone morphogenic protein-7 (BMP-7) single nucleotide polymorphism (SNP) rs230205 [A/G] on susceptibility to hepatocellular carcinoma (HCC) progression from liver cirrhosis after viral hepatitis infection in Egyptian patients. Material and methods: The amplification-refractory mutation system (ARMS)-polymerase chain reaction (PCR) method was used to genotype the rs230205 [A/G] SNP in 150 subjects (50 patients with post-hepatitis C or B cirrhosis, 50 HCC patients, and 50 controls). Expression level of BMP-7 protein was assessed using enzyme-linked immunosorbent assay (ELISA). Results: The results revealed insignificant changes in distribution of all genotypes/alleles of the BMP-7 rs230205 [A/G] SNP between cirrhotic patients, HCC patients and controls. The AA genotype and A allele could be considered risk factors for cirrhosis (OR = 1.75, 1.50) and HCC (OR = 2.19, 1.74), respectively. The AA genotype (95% CI: 0.45-6.79) and A allele (OR = 1.50, 95% CI: 0.77-2.93) may be viewed as cirrhosis risk factors based on group segregation. Additionally, the A allele, AG and AA genotypes and their combined ORs of 2.19 (95% CI: 0.58-8.23), 1.74 (95% CI: 0.90-3.37), and 1.70 (95% CI: 0.68-4.29) could all be risk factors for HCC. No genotype or allele could be regarded as a risk factor for progression of cirrhosis to HCC, according to OR values. Conclusions: The results showed no correlation between BMP-7 rs230205 [A/G] SNP and progression of cirrhosis to HCC. To confirm our findings, additional prospective large-scale research is required.

Genetic variation in FOXP3 and ROR-γ genes in pediatric acute lymphocytic leukemia (ALL) patients: correlation with associated cytokines.

BACKGROUND: FOXP3 and ROR-γ genes are master regulators of the Treg and Th17 differentiation, respectively. This work was planned to investigate the impact of FOXP3 (rs3761548C/A and rs3761549C/T) and ROR-γ (rs9017A/G & rs9826A/G) gene polymorphism on the vulnerability of pediatric Egyptians to acute lymphoblastic leukemia (ALL). Furthermore, we evaluated the impact of these genetic variations on Treg/Th17-related cytokines. METHODS: FOXP3 SNPs were genotyped using PCR-based restriction fragment length polymorphism (PCR-RFLP), while ROR-γ SNPs polymorphism were performed by PCR-sequence-specific primer (PCR-SSP). An Enzyme-linked immunosorbent assay (ELISA) was used to assess the levels of Treg/Th17 associated cytokines on 128 ALL children and 124 healthy donors. RESULTS: Compared to controls, patients had a significant increase (p < 0.01/p < 0.05) in FOXP3rs3761548CC genotype and a significant decrease (p < 0.001/p < 0.01) inrs3761548CA genotype. A significant elevation (p < 0.001/p < 0.01) in ROR-γ rs9017AA genotype and a significant reduction (p < 0.01/p < 0.05) in rs9017AG genotype were detected in ALL patients versus controls. An insignificant change in FOXP3 (rs3761549C/T) and ROR-γ (rs9826A/G) genotypes was demonstrated between both groups. ROR-γ GG and GA haplotypes were significantly decreased (p < 0.05/p < 0.05; p < 0.05/p < 0.05) in ALL subjects compared to healthy ones. Relapsed patients had a significantly higher (p < 0.05/P < 0.05) frequency of FOXP3 rs3761548CA genotype than non-relapsed subjects. ROR-γ rs9017AG and rs9826GG genotypes might be associated with the increase in IL-23 plasma level. CONCLUSIONS: Our preliminary data provided evidence for the impact ofFOXP3 (rs3761548C/A) and ROR-γ (rs9017A/G) gene polymorphisms and the occurrence of ALL in Egyptian children. Another large-scale prospective study should be conducted to validate these findings.

Regulation of CD4+CD25+FOXP3+ cells in Pediatric Acute Lymphoblastic Leukemia (ALL): Implication of cytokines and miRNAs.

Regulatory T cells (Tregs) is one of the immunosuppressive subsets of CD4+ T cells characterized by transcription factor forkhead box protein P3 (FOXP3) expression which are involved in tumor development and progression. Identification of the factors that influence Treg cell function is extremely important. Our current study aimed to evaluate the frequency of Treg cells, cytokine secretion and the expression of microRNAs (miRNAs) in pediatric acute lymphoblastic leukemia (ALL) patients. The frequency of CD3+, CD4+ and CD4+CD25+FOXP3+ Treg was assessed by flow cytometry in 43 ALL patients versus 42 controls. Plasma levels of IL-10, transcription factor ß (TGF-ß), IL-6, IL-17, IL-23 and tumor necrosis factor (TNF-α) were measured by Enzyme-linked immunosorbent assay (ELISA). miR-21, miR-24, miR-26a, miR133b, miR-148a and miR-155 expression were analyzed using quantitative real-time polymerase chain reaction (qRT-PCR). A slight insignificant increase in Treg cells in ALL patients compared to controls was observed. There was a significant elevation in IL-10 (p < 0.05), IL-6 (p < 0.01), IL-23 (p < 0.05) and TNF-α (p < 0.01) in ALL patients compared with controls. Meanwhile, a significant reduction in TGF-ß (p < 0.001) was recorded. A slight insignificant decrease in IL-17 in ALL patients was observed.ALL patients showed a significant increase in miR-21 (p < 0.05), miR-148a (p < 0.01), miR-24 (p < 0.05) and a significant reduction in miR-155 (p < 0.01). In conclusion, the slight change in Treg cells frequency and alteration in related cytokines could possibly involve in the pathogenesis of ALL. Dysregulated miRNAs, as a regulatory mechanism of epigenetics, might contribute to these observed results. Further researches are required to confirm our interesting findings.

Combined Hyperthermia and Radiation Therapy for Treatment of Hepatocellular Carcinoma.

Background: There is no doubt that hyperthermia is one of the powerful radiosensitizers. Finding a proper mechanism working in hyperthermia/radiation combination is still pronounced challenge. Objectives: This study is focusing on the anti-cancer activities (anti-proliferative, anti-angiogenic and antiapoptotic) of thermoradiotherapy. Materials and Methods: Liver cancer cell line (HepG2) was treated by 37oC, 40oC and 43oC hyperthermia degrees combined with three radiation doses (2 Gy, 4 Gy and 8 Gy) for 24, 48 and 72 hrs. Cell viability, apoptotic/necrotic cell screening, apoptotic (BAX and FasL) and antiapoptotic (BCL-2 and GRP78) genes, and pro-angiogenic mediators [vascular endothelial- (VEGF) and Platelet derived-growth factors (PDGF) ware investigated. Results: Our data showed that 40oC temperature combined with 4 Gy radiation gives a significant decrease (p<0.05) in cell viability. Maximum cytotoxicity was reported 48 hr post-treatment followed by slight restoration of cell viability after 72 hr. Compared with untreated cells, only 5% of viable cells with a high percentage of apoptotic (31%) and necrotic (63%) cells were demonstrated in 40oC/4 Gy/48 hr group. Expression of pro-apoptotic genes (BAX and FasL) were increased after hyperthermia with apparent elevation in 40oC/4 Gy/48 hr group coincides with moderate expression of antiapoptotic BCL-2 and GRP78 genes. A significant reduction (p<0.001; p<0.05) in VEGF and PDGF levels; respectively was shown at 40oC/4 Gy/48 hr group. Conclusions: This pilot study proposed 40oC mild temperature hyperthermia as a favorable hyperthermal condition with 4 Gy radiotherapy in HCC treatment. A further research has to be performed considering an application of more than one session of radiothermal therapy at 40oC/4 Gy for total abrogation of cancer cells.

Interleukin-6 promotor gene polymorphisms and susceptibility to chronic hepatitis B virus in Egyptians.

AIM: To investigate the association between IL-6 polymorphisms (-174G/C, -572G/C and -597G/A) and susceptibility to chronic hepatitis B virus (CHB) infection. METHOD: Total 108 subjects with CHB infection and 102 healthy controls were enrolled in this study. IL-6 (-174G/C) was genotyped using Mutagenically separated Polymerase Chain Reaction (MS-PCR) while sequence specific primers-PCR (SSP-PCR) was used for studying -572G/C and -597G/A. IL-6 plasma level was measured using Enzyme-linked immunosorbent assay (ELISA). RESULTS: A significant increase (P < 0.01, P < 0.01, P < 0.001) in -174GG, -572GC and -597GA; respectively in the CHB group compared to control group, while -572GG genotype was significantly decreased (P < 0.01) in CHB patients. A significant increase (p < 0.01, p < 0.01) in -174 G and -597A alleles was observed in the CHB patient group; respectively. GGA haplotype is significantly increased (P < 0.05) while GCA haplotype is significantly decreased (P < 0.001) in the patient group. A moderate linkage disequilibrium (LD) (D' = 0.719, r2 = 0.474; P < 0.001) between IL-6 (-572G/C and -597G/A) was observed. A significant reduction (P < 0.01) in IL-6 plasma level in CHB patients compared to healthy controls (22.28 ±â€¯1.93 versus 32.08 ±â€¯2.41), which was negatively correlated (r = -0.216; P < 0.01) with HBV infection. CONCLUSIONS: This study pointed to the potential role of IL-6 (-174G/C, -572 G/C and -597G/A) gene polymorphisms in the susceptibility to HBV infection. Our results allow for only preliminary conclusions due to relatively small sample size. There is a need for further larger scale studies to fully examine the possible relationship between these cytokine gene polymorphisms and the development of CHB.

Association of TNF-Alpha gene polymorphisms and susceptibility to hepatitis B virus infection in Egyptians.

Tumor necrosis factor alpha (TNF-α) is one of the important cytokine in generating an immune response against hepatitis B virus (HBV). Genetic polymorphisms might influence gene transcription, leading to disturbance in cytokine production. We hypothesized that single nucleotide polymorphism (SNPs) in TNF-α gene could affect the pathogenesis of HBV. To test this hypothesis, we investigated the role of TNF-α polymorphism [-863C/A (rs1800630), -308G/A (rs1800629), -376G/A (rs1800750), -857C/T (rs1799724) and +489G/A (rs1800610)] in the susceptibility to chronic hepatitis B (CHB) infection. Polymorphisms of the TNF-α (-863C/A (rs1800630), -308G/A) were analyzed by Polymerase chain reaction sequence specific primer (PCR-SSP) while TNF-α (-376G/A, -857C/T and +489G/A) by PCR-restriction fragment length polymorphism (PCR-RFLP) in 104 patients with CHB and 104 healthy controls. The plasma level of TNF-α was measured using Enzyme-linked immunosorbent assay (ELISA). The study showed a significant increase in the frequency of -863CC, -376GA, -857CC, -857TT and +489GA genotypes and -863C, -376A, -857C, and +489A alleles in CHB patients compared to controls. In addition, CAGCG haplotype had a highest frequency in CHB patients. A strong Linkage Disequilibrium (LD) between TNF-α -863C/A (rs1800630) and -376G/A (D' = 0.7888, r2 = 0.0200); -308G/A and -857C/T (D' = 0.9213, r2 = 0.1770); -308G/A and +489G/A (D' = 0.9088, r2 = 0.1576) was demonstrated. CHB patients had significantly lower levels of TNF-α compared to controls. In conclusion, our preliminary results suggest that -863C/A (rs1800630), -308G/A, -376G/A, and +489G/A of the TNF-α gene may play a role in HBV susceptibility in Egyptians. The significant reduction in TNF-α in CHB patient was independent of any particular genotype/haplotype in TNF-α.

Hepatitis C Virus Associations with Non Hodgkin's Lymphoma: Insights on Inflammation/Angiogenesis and CD Markers.

We aimed to investigate any association between hepatitis C virus (HCV) infection and non-Hodgkin's lymphoma (NHL) in the view of cytokines that control inflammation/angiogenesis and their correlation with certain CD markers. NHL patients with or without HCV infection were studied. CD5, CD30, CD3, CD20 and CD45 were immunohistochemically evaluated. Plasma levels of vascular endothelial and platelet derived growth factors (VEGF, and PDGF), tumor necrosis factor (TNF-α), transforming growth factor (TGF-ß), interleukin-6 (IL-6), IL-8, IL-4, IL-12 and interferon gamma (IFN-γ) were detected by enzyme-linked immunosorbent assay (ELISA). HCV+ve NHL patients showed a significant reduction in VEGF, PDGF, IFN-γ, CD5 and CD45 and a significant increase in IL-12 and IL-8. In conclusion, there was a significant change in cytokine secretion and expression of CD markers in HCV+ve NHL patients. Based on our results, HCV infection in NHL patients requires more in-depth investigations to explore any role in lymphoma progression.

CD38 and interleukin 6 gene polymorphism in egyptians with diffuse large B-cell lymphoma (DLBCL).

Given the importance of understanding the genetic variations involved in the pathogenesis of non-Hodgkin's lymphoma (NHL), this pilot study was designed to investigate the impact of CD38 (184C/G; rs6449182) and IL-6 (-174 G/C; rs1800795) gene polymorphism on susceptibility of Egyptians to diffuse large B cell lymphoma (DLBCL); major types of NHL. To the best of our knowledge, this study is the first one that examines CD38 polymorphism in the NHL. Genotyping polymorphism is performed using restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) for CD38 and Mutagenically separated PCR (MS-PCR) for IL-6 in 100 Egyptian NHL patients with DLBCL subtype and 119 normal controls. The serum level of IL-6 was measured using Enzyme-linked immunosorbent assay (ELISA). CD38 (184C/G) genotype is significantly increased in NHL patients (p < 0.01), while the GG genotype is significantly increased in controls (p < 0.05). Only two genotypes were found (GG and GC) in IL-6 (-174), no CC in our NHL patients and only one case in the controls. Insignificant change in IL-6 (-174 G/C) genotypes was recorded. Significantly increased serum IL-6 (p < 0.05) was positively correlated (r = 0.17; p < 0.05) with the disease. Taken together, our data stressed the importance of CD38 gene polymorphism in developing DLBCL. Our pilot study indicates that CD38 (184) CG genotype might play a role in DLBCL susceptibility in Egyptians. Additional prospective studies on larger population are needed to confirm our findings.