3'UTR enhances hCD47 cell surface expression, self-signal function, and reduces ER stress in porcine fibroblasts.

INTRODUCTION: Macrophages contribute to xenograft rejection by direct cytotoxicity and by amplifying T cell-mediated immune responses. It has been shown that transgenic expression of hCD47 protects porcine cells from human macrophages by restoring the CD47-SIRPα self-recognition signal. It has also been reported that the long 3' untranslated region (3'UTR) of the hCD47 gene, which is missing from constructs previously used to make hCD47 transgenic pigs, is critical for efficient cell surface expression in human cells. The aim of this study was to investigate the impact of a modified form of the 3'UTR on the expression, localization, and function of hCD47 in transfected porcine cells. METHODS: hCD47 constructs with and without the modified 3'UTR were knocked into the GGTA1 locus in porcine fetal fibroblasts using CRISPR. Flow cytometry of the transfected cells was used to analyze hCD47 localization. Endoplasmic reticulum (ER), mitochondrial, and oxidative stress were examined by gene expression analysis and confocal microscopy. Phagocytosis of transfected cells by human macrophages was measured by flow cytometry, and stimulation of human/non-human (NHP) primate lymphocytes by the cells was examined using a PBMCs proliferation assay. RESULTS: Cells transfected with the construct lacking the 3'UTR (hCD47(3'UTR-)) exhibited predominantly intracellular expression of hCD47, and showed evidence of ER stress, dysregulated mitochondrial biogenesis, oxidative stress, and autophagy. Inclusion of the 3'UTR (hCD47(3'UTR+)) decreased intracellular expression of hCD47 by 36% and increased cell surface expression by 53%. This was associated with a significant reduction in cellular stress markers and a higher level of protection from phagocytosis by human macrophages. Furthermore, hCD47(3'UTR+) porcine cells stimulated significantly less proliferation of human/NHP T cells than hCD47(3'UTR-) cells. CONCLUSION: Our results suggest the potential benefits of using hCD47 constructs containing the 3'UTR to generate genetically engineered hCD47-expressing donor pigs.

Wide genetic variations at 18S ribosomal RNA locus of Cyclospora cayetanensis isolated from Egyptian patients using high resolution melting curve.

A variable clinical picture of cyclosporiasis including gastrointestinal tract (GIT) symptomatic or asymptomatic beside extraintestinal consequences suggests a possibility of heterogenicity of Cyclospora cayetanensis. The present work aimed to explore the possibility of genetic variation of C. cayetanensis using high-resolution melting (HRM) curve of polymerase chain reaction (PCR) amplified 18S rRNA genes. DNAs extracted from the stool samples of 70 cyclosporiasis patients were amplified and scanned by PCR/HRM curve. The results showed that there are four different genotypic profiles of C. cayetanensis with presence of mixed ones. Although Tm of all profiles was within the same range, they were discerned by plotting of the temperature-shifted florescence difference between normalized melting curves (dF/dT). Genotypic profile I was found alone in 40 % of patients and mixed with genotypic profile II and/or III in 25.7 % of patients, followed by genotypic profile II in 14.3 % then genotypic profile III and IV (10 % each). A significant relation was found between genotypic profiles and GIT symptomatic status as profile I and profile II were mostly detected in patients with acute GIT symptoms without or with chronic illness, respectively, while profile IV cases only were GIT asymptomatic. Statistical significance relations between genotypic profiles and age, gender, residence and oocyst shape index were determined. In conclusion, PCR/HRM proved a wide variation on C. cayetanensis genes that could be reflected on its pathogenic effects and explaining the variability of the clinical manifestations presented by cyclosporiasis patients.

Evaluation of circulating Transforming growth factor-beta1, Glypican-3 and Golgi protein-73 mRNAs expression as predictive markers for hepatocellular carcinoma in Egyptian patients.

Hepatocellular carcinoma (HCC) incidence is fast-growing especially in countries highly prevalent with viral hepatitis. Its poor prognosis has driven the research toward the discovery of sensitive markers for early detection. We investigated the usefulness of serum Transforming growth factor-beta1 (TGF-ß1), Glypican-3 (GPC3), and Golgi protein-73 (GP73) mRNAs as early biomarkers in HCC Egyptian patients chronically infected with hepatitis C virus (HCV) in comparison with serum alpha-fetoprotein (AFP). Using semi-quantitative RT-PCR and densitometry analysis, circulating TGF-ß1, GPC3, and GP73 mRNAs expressions were estimated in 15 healthy adults, 15 chronic HCV (CHC) patients and 25 HCC patients. Serum GP73 expression percentage in HCC group was significantly higher than controls (100 vs. 40 %, P ≤ 0.001) and when compared to elevated serum AFP levels (100 vs. 36 %, P ≤ 0.001). TGF-ß1 and GP73 expression means were also higher in HCC patients than controls and CHC patients (P < 0.05). GPC3 expression showed higher frequency in CHC patients compared to HCC group (80 vs. 28 %, P = 0.0016). According to the study cutoffs, serum TGF-ß1 and GP73 mRNAs showed 60 and 96 % sensitivities for HCC diagnosis with 100 and 95 % specificities, respectively. Furthermore, elevated GP73 mRNA expression levels in early HCC were significantly increased compared to those of TGF-ß1 mRNA and to high serum AFP (92.3 vs. 53.8 and 23.1 %; P = 0.03 and 0.0004, respectively). In conclusion, circulating TGF-ß1 and GP73 mRNAs could be useful biomarkers for HCV-induced HCC diagnosis. Moreover, serum GP73 mRNA is sensitive for early cancer detection than AFP and TGF-ß1 mRNA. However, these results need further validation studies.

Helicobacter Pylori DNA in Liver Tissues From Chronic Hepatitis C Egyptian Patients.

BACKGROUND: Hepatitis C virus (HCV) is considered the most common etiology of chronic liver disease in Egypt, which may progress to cirrhosis and hepatocellular carcinoma (HCC). Previous studies have documented an association between Helicobacter pylori (H. pylori) infection and liver cirrhosis with or without HCC. This study aimed to investigate the presence of H. pylori DNA in the liver tissue of Egyptian patients with chronic hepatitis C (CHC). METHODS: Fifty-two CHC Egyptian patients were enrolled in this study. Plasma anti-H. pylori IgG was assessed with ELISA. Liver biopsies were tested for presence of Helicobacter DNA using genus specific nested polymerase chain reaction (PCR) and species was identified by sequencing. RESULTS: Anti-H. pylori IgG was detected in 31/52 (59.6%) CHC patients while Helicobacter DNA was detected in 6 (11.5%) patients, all were H. Pylori by sequencing. Helicobacter DNA was more frequent in patients with high stage liver fibrosis (33.3%) than in those with low stage fibrosis (2.7%) (P = 0.006). There was no association between the presence of H. pylori DNA in the liver and age, gender of patients, liver function tests, AFP levels or viral load. CONCLUSIONS: These data confirm the presence of H. pylori DNA in liver of some CHC Egyptian patients and suggest an association of this bacterium with progression of liver fibrosis.

Serum interleukin-8 and insulin like growth factor-1 in Egyptian bladder cancer patients.

BACKGROUND: Bladder cancer is a major health problem in Egypt as it represents the most common malignancy. AIM: Evaluation of the potential usefulness of serum IL-8 and IGF-1 in Egyptian bladder cancer patients. METHODS: Serum levels of IL-8 and IGF-1 were determined in 51 bladder cancer patients and 45 controls using a chemiluminescence enzyme immunometric assay. RESULTS: Serum IL-8 was significantly higher in cancer patients than in controls (P < 0.0001). It was significantly higher in patients with invasive cancer than those with superficial cancer (P < 0.01). Also, IL-8 showed a significant elevation in relation to schistosomal infection (P = 0.02) however, it did not differ in relation to either pathological type of tumor or its grade (P > 0.05). Receiver operating characteristic (ROC) curve indicated that IL-8 cut-off value of 35 pg/mL yielded the best combination of sensitivity (71%) and specificity (98%) for differentiating patients with bladder cancer from control subjects. Serum IGF-1 levels showed no significant difference between bladder cancer patients and controls (P > 0.05). There was no relationship between IGF-1 levels and clinicopathological parameters. CONCLUSIONS: In Egyptian patients with bladder cancer, serum IL-8 is significantly elevated and its level is related to tumor invasion and associated schistosomal infection. Moreover, serum IGF-1 level does not help as a serum tumor marker in these patients.

Expression of telomerase reverse transcriptase in psoriatic lesional skin.

BACKGROUND: Little information is known about telomerase expression in the chronic benign hyperproliferative skin disease, psoriasis. Further studies are still required to investigate its usefulness as a biomarker of this skin disorder. AIM: To investigate the expression of human telomerase reverse transcriptase (hTERT) in psoriatic lesional skin and its relation to disease severity. METHODS: The levels of hTERT-mRNA were quantified using real time RT-PCR in lesional versus nonlesional skin specimens from 24 psoriatic patients. RESULTS: The expression of hTERT was detected in 16 psoriatic lesional specimens (66.7%), but in none of the normal skin. There was no relation between hTERT expression level and age of the patient or the duration of the disease. Among hTERT-positive patients, a significant positive correlation was observed between hTERT-mRNA levels and both the Psoriasis Area-and-Severity Index (PASI) and scaling scores (p = 0.012 & p = 0.006, respectively). CONCLUSION: Telomerase mRNA is detectable in lesional skin of most psoriatic patients and correlates with the severity of the disease and the rate of epidermal proliferation.